The Archmage in GrimGrimoire. The moment he stops being mentioned as being a defeated threat and starts being an active character, things get a lot more serious, fast, and what was once a light-hearted magical school drama becomes much darker.
Hawke in Advance Wars 2 manages to simultaneously pull off a Knight of Cerebus and a Worthy Opponent. While the game's Quirky Miniboss Squad are about as quirky as you can get without major surgery, Hawke is serious to a fault, especially when facing off against Eagle: when his giant sea fortress comes under attack by Eagle's squadron is approaching, he says "how predictable" and surrounds it with anti-air units, forcing the player to resort to clever tactics and massive casualties. And later on, he decides to stop Eagle from interfering all together by forcing his troops to march past a place where his air units can't go: an active volcano.
The entire Bolt Guard from Advance Wars Dual Strike manage to one-up Hawke big time. While other games were cartoonish and out for world domination, Von Bolt and his Bolt Guard intend to drain the very life of the Earth itself, leaving the entire planet a lifeless husk of desert and sand, and unleash the Oozium 223 that are graphically described by Kindle to "melt the flesh right off their victims". It's really telling that it's enough to make the former baddies, Hawke and Lash, question if it's even worth being that evil, which gets them marked for death and forces them to flee to the good guys for survival.
Then we have Dr. Caulder from Advance Wars Days Of Ruin, whose plans involve unleashing a horrible virus that causes plants to erupt from beneath your skin, give weapons to both sides of a war to increase casualties, face people with horrific choices, perform experiments on people, murder the survivors of an apocalypse, and destroy factories capable of manufacturing food and water...with absolutely no motive outside of seeing "what would happen".
Grand Theft Auto: San Andreas is largely a Black Comedy with most less than moral actions the protagonist CJ or the other criminals commit being treated humorously and most the characters being extremely quirky. The exceptions to this involve the game's Big Bad, Frank Tenpenny. The scenes with him are almost entirely humorless, any humor involving him is mostly played to show how much of Jerkass he is, along with the rest of his personality, and unlike the other characters, he shows that he has no absolutely no sense of loyalty to others and flat scoffs on the idea.
Dimitri Rascalov from Grand Theft Auto IV. After revealing his true colors in "Russian Revolution", he definitely took the storyline in a darker direction. A meta example by the fact that he is a disturbingly straight example, and his cruelty is played in the darkest way possible.
Darko Brevic as well. His only appearance in "That Special Someone" is perhaps one of the darkest and most dramatic moments in the game.
Kurtis looked like one of these in the first game; up until that point, Laharl's major enemies (Unknown Rival Mid-Boss, Love Freak Flonne, Dirty Coward Madeiras and Lord Error-ProneCaptain Gordon, Defender of Earth!) had been little more than comedic interludes, to which the cyborg proved to be remarkably strait-laced by comparison. It turned out he was neither particularly evil nor particularly competent—the real Knight of Cerebus was his boss, General Carter. Who had, it later turns out, made a deal with Vulcanus (who was being manipulated by Seraph Lamington)!
There's also Judge Nemo in the fourth game, or real Overlord Zenon in the second game in this regard. The first time the latter is introduced, she defeats Laharl which had been previously a Hopeless Bossfight. The second time is by the end, in the case you did everything right, unlocks an infamous Downer Ending.
Xenolith in Disgaea Dimension 2. Even before he's revealed as the Big Bad, there's nothing comedic about him, in a sharp contrast to the usual Disgaea humor.
Malice in Riviera: The Promised Land. Her first appearance is part of a very dark scenario, but the game returns to its lighthearted self when she leaves. Her return marks the shift of the game to its main themes and the serious core of the series.
The LEGO Adaptation Game series is a barrel of laughs and fun to play, although every now and then there is a villainous exception.
In LEGO Star Wars, the Emperor doesn't abide by this rule; he rarely has any funny scenes to show, minus one with his alter ego and he's more or less played seriously in comparison to the rest of the games.
LEGO Harry Potter has an odd aversion of this trope with Voldemort, who ends up more like an Ineffectual Sympathetic Villain (at least until Years 5-7 came out). It gets played straight with Umbridge, who's almost as cruel as her book and film counterparts.
In his brief screen time in his physical form in Lego Lord of the Rings, Sauron is this, being presented as every bit the terrifying menace as in the scene in Fellowship of the Ring. And in this version, he's portrayed as a Bionicle figure and is at least 5 times larger than the rest of his soldiers. Similarly, despite their mild doses of Adaptational Comic Relief, the Nazgul are almost every bit as scary as the others.
Lego Marvel Super Heroes is again mostly funny, although there are a couple of weird examples. When Venom shows up, the level you're in suddenly ends up with a Survival Horror feel to it, although he is subdued early on. The best example, however, would be Loki; while not entirely without his funny moments, mostly his childishness in terms of his attitude to Thor, by the end of the game he almost comes across as an even more unnerving, dangerous sociopath than his movie version. Loki's plan is to unleash Galactus upon Earth and Asgard as part of his petty revenge, so that both realms will be utterly obliterated. This is not played for laughs, needless to say.
LEGO Batman 3: Beyond Gotham has Brainiac. While not without his comical moments much in the way that Lex does, his introduction ends up shifting the genre from typical superhero/supervillain conflict to intergalactic crisis, overshadowing Lex and the Joker and shrinking the Earth. Moments where he mind-controls the heroes stand out as among the darkest in the entire franchise, in particular when he brainwashes Batman and nearly kills Robin, or when he enlarges and brainwashes Superman, very nearly crushing Batman onscreen. Aside from Brainiac, the level with Indigo-1 is among the darker moments of the campaign, since it shows her sociopathic nature when the Heel–Face Brainwashing fails, and very heavily implies that she's murdered Abin Sur's daughter as in the comics.
LEGO Dimensions: The Cybermen and Weeping Angels, who are just as frightening here as they are in their home series, with few humorous moments, if any. It's quite jarring, seeing them alongside villains like The Joker, GlaDOS, and Lord Business. The playable Cyberman is toned down a bit, but still has a degree of uncanniness to it.
Final Fantasy has just about every antagonist from IV on, with some being more effective than others.
In Final Fantasy IV, things typically get darker whenever Golbez or the Four Fiends walk in on the scene. The game is a bit light-hearted normally, but whenever they show up, expect the body count to rise.
Final Fantasy V has Exdeath, whose appearances made the game's tone become incredibly serious. In a game like Final Fantasy V, which is very lighthearted and at points silly compared to its predecessors (and many successors), Exdeath's appearance always seemed to stop things flat and ram into the player the severity of their situation.
Final Fantasy VI is a generally light blend of mild angst and whimsical humor...up until Kefka usurps the power of the Warring Triad and causes the apocalypse, creating the World of Ruin and becoming a god. The tone of the game completely changes after, focus shifting to the characters trying to rebuild their ranks and find reasons to continue living in a barren and dying world. Kefka is a very unique case of this trope in that he manages to be amusing as well as vicious. Of course, that all begins to change once he causes aforementioned apocalypse.
Final Fantasy VII has Sephiroth and Jenova. Normally, the game is a light-hearted mixture of humor, Good Bad Bugs, and a "Blind Idiot" Translation to boot ("This guy are sick."). When Sephiroth (actually Jenova disguised as him) shows up, things take a turn for the darker (albeit with one poorly translated exception), where even party members aren't safe. But when you make it to the Northern Cave and meet Sephiroth in person, the game never regains its light-hearted tone.
Final Fantasy XV gives us its Big Bad, Ardyn Izunia. For the majority of the game, he actually helps Noctis and his friends progress through their journey at certain points, acting chummy towards them the whole time (though they don't fall for it and suspect that he's up to something). Come Chapter 9, however, he drops the act and cements his status as a Knight of Cerebus via fatally stabbing Luna while Noctis is helpless to do anything about it before going on to put his true plan into motion. After that, the game's story takes a much darker and more serious turn.
The Paper Mario series has a higher-than-average number compared to the Mario games overall:
Paper Mario: The Thousand-Year Door has Sir Grodus, the leader of the Secret Society of X-Nauts. The rest of the X-Nauts have plenty of humorous qualities (particularly Lord Crump), but he doesn't. While his villainy doesn't initially get much worse than bossing around his minions in a no nonsense manner, he crosses the line into Knight Of Cerberus mode when he has TEC shut down for allying with Peach. Then when he is cornered by Mario and his partners, Grodus threatens to have Peach killed if they try to resist as he blasts them with lightning; he would have killed them right there if Bowser hadn't fallen on top of him. But even his menace is eclipsed by the Shadow Queen, the demon he is trying to free. In the backstory, she had destroyed many towns and murdered thousands while building torture chambers where her prisoners were eaten by her dragons, and created the Crystal Stars to help solidify her reign over the world. She also cursed the four heroes who beat her the first time to be trapped in chests. In the finale, she possesses Peach to be her new physical vessel.
Super Paper Mario has Dimentio. He starts off as exhibiting some humorous personality quirks, much like his fellow minions of Count Bleck. But as the plot progresses, he is revealed to be utterly psychotic, and quickly makes history by becoming the first villain to successfully kill Mario & Luigi (they get better though, as expected). This culminates in him stealing the Chaos Heart from Bleck and planning to remake the multiverse in his image once the old one has been destroyed, and then, when he is defeated, deciding to destroy it all anyway out of spite.
Even the Mario & Luigi games, despite being much wackier than the Paper Mario games, manage to portray a few sinister villains:
For the most part, the villains of Mario & Luigi: Bowser's Inside Story are rather silly. From the eccentric Fawful and his odd speech patterns, to his also eccentric minion Midbus with his goofy rivalry with Bowser, to the brainwashed Koopa Troop and their over-the-top glorification of Fawful, everyone's a source of laughter. Then you get to the Dark Star, the entity Fawful is trying to awaken. Its backstory had it nearly destroying the Mushroom Kingdom before it was sealed up by the Star Sprites, it chokes the Mario Bros. when they get too close to the miasma enveloping it, and when it gets dialogue, it is devoid of humor or any other attribute other than an impulse to destroy everything.
Luigi's Mansion: Dark Moon has put King Boo here. While initially a Well-Intentioned Extremist out to avenge his fellow Boos, his defeat and imprisonment at the end of the first game turned him into a sociopath willing to do anything in order to take down Luigi and Gadd, to the point of kidnapping Mario and trapping him in a painting, planning to do the same to everyone Luigi and Gadd care about, and if that doesn't work, destroy the fabric of the universe by unleashing a massive army of ghosts, causing either a Class X-4 or Z apocalypse.
While Shin Megami Tensei is not a very lighthearted series of games to begin with, they all have their characters that really cause hell to start happening. For example, even when Alraune kidnapped men and a fellow Samurai in the beginning of Shin Megami Tensei IV, it was a bloodless event and nobody died. But shortly after, Kiccigiori burns and you encounter the Black Samurai. As soon as the Black Samurai appears, Mikado begins to crumble apart, 1500 Year old traditions are broken to capture the Black Samurai and this causes the old war of Law and Chaos to start up again.
Shovel Knight: Even after The Reveal, The Enchantress is the only villain in the game who is not played for laughs and is portrayed as evil throughout.
Every game in the Ace Attorney series has one of these near the end.
Justice for All: Hello Matt Engarde. The Ace Attorney series will forever be comedic, and it seems to believe very much in well-earned happy endings, but Phoenix's first truly evil client gave it a definite darker twist that never really left. He also started the trend of having sociopathic villains as the Big Bad.
Trials and Tribulations: An odd case here in Dahlia Hawthorne. The villainess of three of the game's five cases, Dahlia is found guilty of murder in the very first one. However, the true depths of her depravity isn't truly revealed until you see what crimes preceded and succeeded that one.
Apollo Justice: Kristoph Gavin is the villain of both the game's first and last case. And his murder in the first is nothing compared to what he did to Phoenix and the Misham family. For a game whose other two killers are a none-too-smart Bitch in Sheep's Clothing and a goofy-haired rocker, Kristoph's ruthlessness and intelligence is all the more threatening.
Dual Destinies has The Phantom. The Phantom is a mercenary spy so well versed at disguise that even they don't know who they really are. They can be anyone, and for the whole game they've been posing as the Plucky Comic Relief Detective Fullbright, having killed him and stolen his identity years prior. Before the game even starts, the Phantom kills Athena's mother, framing Simon for it and putting him on death row, and nearly blows up a space shuttle, traumatizing the pilot for life. They inadvertently start the Dark Age of the Law due to their actions, indirectly leading to the death of a teacher at a law school. During the game, they blow up a courtroom full of people (who managed to escape luckily) to get rid of one piece of evidence, tried to blow up a shuttle again, and killed Apollo's best friend then framed Athena for it to get her out of the picture. And the worst part? They don't feel any regret for this. In fact they don't feel anything at all. The Phantom is a high functioning sociopath, willing to do anything and kill anyone if their employer says so or to protect their identity.
Investigations: Super-serious and super-dangerous international criminal syndicate leader Quercus Alba who is responsible for just about all of the game's events.
Investigations 2 has Bansai Ichiyanagi. Unlike the killers of the game's previous three cases, he has absolutely no redeeming features whatsoever, his Amoral Attorney streak rivals or even surpasses von Karma (he auctions off evidence from past cases, for one) and he manages to drives Edgeworth into a Heroic B.S.O.D. so bad he resigns as a prosecutor! And just when you think it can't possibly get any worse, he crosses the Moral Event Horizonright in front of everyone by telling his own son, in the cruelest way possible, that he would never have amounted to anything without his help. It's no wonder that he causes until-then rival Hakari Mikagami to go Heel–Face Turn. What's most surprising is the final case's killer actually doesn't fit this trope. In fact, he's a Woobie, Destroyer of Worlds with a Dark and Troubled Past so horrible you can't help but sympathise with him. Still, he can still be pretty creepy.
Persona 3: Shinjiro's arrival marks a huge, sinister change in the atmosphere, especially in the female route in P 3 P because his social link contains a lot of dark foreshadowing. And he dies after one month, sparking Akihiko and Ken's Character Development and showing you, finally, that a game with the tagline "Memento Mori" means exactly what it's saying, and will not pull its punches.
All the Shadow bosses in Persona 4 have some comedic traits to take the edge off their disturbingness, until you get to Shadow Teddie, who is played dead seriously and looks like something out of a horror game. From this point on the game's bosses get a lot darker (with the possible exception of Shadow Naoto) and the murder mystery plot gets a lot more intense.
Shadow the Hedgehog's introduction into franchise took the series down a much darker and grimmer path. Sonic Adventure had a larger share of dark moments than any of the previous games but ultimately contained an upbeat tone. Sonic Adventure 2 (Shadow's debut) is certifiably a point at which the series started experimenting with much heavier themes and Shadow was certainly a catalyst in the series' move in that direction (which continued all the way up to Sonic Unleashed).
And he also gets beaten out by Mephiles the Dark in Sonic the Hedgehog (2006), who was depicted as having no justifications behind his horrific actions other than enjoying destroying things simply because he could, succeeded in murdering one of the heroes, and even successfully shattered the fabric of existence until he was defeated. He doesn't just get killed, he gets completely erased.
And prior to Shadow the Hedgehog's introduction, there was Chaos from Sonic Adventure. Even though Sonic Adventure was much lighter in tone than Sonic Adventure 2, Chaos' introduction introduced some darker themes into the Sonic the Hedgehog franchise, such as having a dark backstory where the Echidna civilization was wiped away by Chaos, which was pretty dark for the Sonic the Hedgehog franchise at the time.
Black Doom from Shadow the Hedgehog is a pretty dark villain as well, having his aliens invade the world and planning to kill all humans (though he claims to only be trying to save them from a path of self-destruction). If Shadow chooses to help Black Doom in the Pure Dark path (and the player must view every ending in order to unlock the real ending) Black Doom has Shadow blow up a city with a bomb. Also, in the real ending of the game, Black Doom paralyzes Sonic, Tails, Knuckles, Amy, Rouge, and Eggman and tries to feed them to carnivorous aliens.
The Deadly Six in Sonic Lost World are an odd case: while they are somewhat goofy, hilarious, brightly colored characters, they prove to be some of the darkest villains in the series: upon betraying Eggman, they use one of his machines to drain the planet below of its energy (which is implied to be its life force) to make themselves stronger. Then, they plan to turn Sonic into a loyal robot, but due to a rescue by Tails, they capture him instead. Not that they don't try to turn him into a robot anyway, however, and after they have him, Sonic stops making fun of them and demands they give him back, unharmed. Eventually, Amy and Knuckles get their lives drained by the machine, and they (seemingly) manage to kill Eggman. It should be noted that this is the first time in the series where the events of the story actually manage to briefly break Sonic.
In the first game, the murderous animatronics all at least have some humor potential (in a Black Comedy way). All except for Golden Freddy, an Easter Egg in the first game, and the most lethal enemy in the second game. Nothing about him is funny, not his appearance, not his powers, not the true role he may play in the story. NOTHING.
Five Nights at Freddy's 3 has Springtrap. Not only is his appearance absolutely horrifying compared to the other animatronics (yes, even Golden Freddy), but he's actually the murderer, having been crushed to death in what is implied to be the hybrid suit he used to perform his murders. And sometimes you get a view of exactly what's inside him during the game's opening on occasion.note It's the corpse of the murderer.
Pokémon Mystery Dungeon: Gates to Infinity ups the ante with the introduction of Kyurem, a grim, nihilistic No-Nonsense Nemesis who is dead set on seeing the Bittercold, an Eldritch Abomination made out of the collective suffering of the setting's Crapsack World, destroy the world - himself included. To this end, it's revealed Kyurem has killed dozens of humans sent to the Pokemon world sent to stop the Bittercold and defines himself in his second appearance by killing Hydreigon and beating the hero to a pulp without saying a word. Nothing about the character is played lightly in the slightest, and Kyurem may actually be an even more effective example of Darkrai due to both an explicit body count and a motive and character that extends past a standard two-dimensional Omnicidal Maniac.
Pokémon Super Mystery Dungeon started out as a Slice of Life-adventure story but then came the second half in which right after the heroes found the petrified Latios and Latias, Entei shows up and threatens to kill the protagonists on the spot. While he isn't strictly villainous, any scene that centers on Entei means a flip of the off switch on the franchise's lighthearted nature and presents a clear sense of danger. Later on, Nuzleaf and Yveltal pick the torch from Entei by turning almost everyone in the world into stone.
Ghetsis from Pokémon Black and White. Would you like some horrific implied child abuse and attempted murder with your cute monster-battling game?
Colosseum and XD represents this for the franchise at large, as not only is the Orre region Darker and Edgier than the rest of the known world, the Cipher syndicate is much nastier than anything that came before them. Corrupting Pokémon via Mind Rape and nearly blowing an entire island with several Admins and the player on it are just a couple of things they've done. The Sequel Hook after XD does not help, and neither does the implications that Ghetsis' plot would have left Unova ripe for Cipher to conquer. In any of the other games' WMG sections, bringing Cipher up darkens the discussion in record time.
Purple Eyes from Pokémon Ranger Guardian Signs fits this trope to a T. He's beaten Rand badly and kidnapped his wife and daughter, outright attempts murder (which, mind you, has only been attempted by a few Pokémon villains such as a certain Dragon from Shadows of Almia and the likes of the Pokémon Mystery Dungeon villains) ranging from you and your friends, to the entire region, to the entire human population except himself, he mugs the elderly Societea members (who deserved it, but still), laughs after making Mewtwo attack Edward and Rand, laugh once again at the thought of everyone in Oblivia being killed, as mentioned before, after being broken by questing at the Ranger Union, he turns into a Misanthrope Supreme, tries to convince ARCEUS to kill all humans except himself and let him rule under him. After a battle with Arceus, he deems it unsafe for him to be in the mortal plane and whisks him off to his world/dimension, to be judged. And it looks how he might possibly be related to Sabios, the big bad from the past missions, and that he at least looks like he's 21. And there's no knowing what this guy has done before he became a member of the Pinchers. There's a reason this guy's entry is spoilertastic.
Lysandre from Pokémon X and Y is a Fallen HeroOmnicidal Maniac determined to kill everyone, people and Pokémon alike, who is not a part of Team Flare. Unlike Cyrus, he has no plans to make a new world; he just wants to wipe everything out. And he nearly succeeds. He's also the only villain to be seemingly Killed Off for Real in the main game series.
Cyrus himself. Cyrus is an unusual example in that every following villain has seemingly been designed to top him, with the silliness removed. Whereas Team Rocket were Only in It for the Money, and Team Aqua/Magma were both Well-Intentioned Extremist groups, Cyrus... is a Well-Intentioned Extremist in that he is an Omnicidal Maniac that seeks to remake the world so that it is without emotion or spirit. He's also implied to have a dark and horrible backstory (with some explicit reference to his parents being emotionally abusive at best), and, to date, is the only main game antagonist to be a Karma Houdini, with a horrifying Sequel Hook in that he is still in the Distortion World, working to unlock its secrets.
Kyogre and Groudon. The real villains are comical and misguided, even willing to absolve for their sins, but things go south very quickly once they summon Groudon or Kyogre. Kyogre and Groudon pose a threat to the entire world once they appear because their powers can affect the weather, causing an intense heat wave that could burn the world (Groudon) or flooding it with an endless rainstorm (Kyogre). The sequence that takes place during their rampage is one of the most chilling in the franchise, especially in the remakes. There's a good reason that the villainous team leaders seriously regret their choices shortly after Kyogre/Groudon's emergence.
Continuing the tradition, in Pokémon Sun and Moon, the true villain, Aether Foundation President Lusamine, is among the darkest villains in the franchise, period. She's an abusive mother extraordinaire who treats her own children as objects to be discarded — going as far as to try and directly murder one of them, placing her a head above even Ghetsis — and is willing to wreak wide swathes of destruction across Alola due to her psychotic obsession with the eldritch Ultra Beasts (which are heavily implied to have driven her insane after her husband vanished into Ultra Space, to boot). Lusamine goes as far as to physically fuse with one of these Ultra Beasts, and the ensuing battle is likely among the most disturbing in the franchise. She's earned a net of comparisons to Ragyo Kiryuin for good reason.
The Ultra Beasts also spark a darker tone once they appear, with much being made of their destructive power, violent tendency and threat to both humans and Pokémon. Their postgame storyline is among the darkest plots in the series, with the implication that the International Police are using the player (who's no more than eleven) as human bait, as well as Looker telling them that if they can't catch the Ultra Beasts, they must be destroyed (not that the player is even given the option to destroy them). Even within this storyline, there's a Knight in the form of UB-05: Glutton, a.k.a. Guzzlord. It's revealed that ten years prior, a Faller used as bait was "done in" by it as a result of Looker taking pity on it. Rarely if ever has a Pokémon outright killing a human been mentioned before.
Galacta Knight is revealed to be this in Kirby Super Star Ultra. He destroyed entire civilizations, as shown in flashbacks, and, according to Nova, whom Meta Knight made his wish to fight the greatest warrior in the galaxy to, was so dangerous that he was sealed away out of fear that his power was too great.
Chaos Lord Ledgermayne. Basically, many villains before it, especially some of the previous Chaos Lords, were lighthearted and comical in nature, and even mainly focused on Incredibly Lame Puns, big deal. Then cue the arrival of Ledgermayne, who proves to be almost invincible due to being immune to regular weapons and magics and even being able to control magic itself. And later, Ledgermayne reveals its plan to cut off all magic from Lore - all without caring about the fact that all life on Lore will die if it itself succeeds, which it is, of course, fully aware of.
Vordred, proves to be this as well. He shows players that he means business by using his signature spell, the Voiduminance Necrot-Morph, to turn other people, especially the very paladins he was trained to fight and destroy (after all, he is a Paladinslayer), into his undead slaves. And that's not all, his armor, which is made up of Too Many Skulls, is immune to light-based magic, and he gets even more powerful thanks to an experiment performed on him by ArcAttack with the help of the hero, plus he's the reason why Part 1 of the Doomwood saga is Darker and Edgier than the previous sagas in the game before it.
Bloodtusk Ravine's story proves to be the darkest out of all the Chaos Lord areas so far, seeing how Xing & Xang's scheme for the ravine is darker than their previous schemes were, and Krellenos also lands himself in this spot since he worked behind the scenes during the war between the Horcs and the Trolls and even murdered his own brother Antiphuus. Then Khasaanda kills and usurps her own twin brother, planning to use his powers to exact revenge on Drakath for what happened to her brothers themselves.
Sepulchure himself in the games made by Artix Entertainment that he appears in fits, seeing as how there's less humor when he's around. In contrast with many Harmless Villains working for him, he invokes fear in others and kills off many people, including the ones in the Guardian Tower he crashed his fortress into, as a show of proving that he means business. Of course, his lack of remorse in turning Fluffy into a Dracolich and causing death and destruction doesn't stop him from being an Anti-Villain with standards who loves his daughter Gravelyn so much that he becomes unwilling to kill her despite her being the Champion of Light he sought to destroy, and is trying to bring back his lost love Lynaria.
Hades and, to a far greater extent, the Chaos Kin in Kid Icarus: Uprising. Hades is very funny, but he still happily cartwheels over every line he can find just for fun and the sheer fact that he's an asshole. The Chaos Kin doesn't even have that, destroying for no reason and being the only character in the game who is taken completely seriously.
The Legend of Zelda: Skyward Sword takes a somewhat more balanced approach with the franchise, using dark elements but also lighter moments within the same game, and also lampshading the series' traits. Ghirahim is a notable example of this, as he still comes off as intimidating despite his... interestingbehavior. The game will usually take a turn for the serious whenever he appears. His owner, Demise, isn't much better.
Porky in MOTHER 3 also counts, being the one behind all the horrible events taking place over the course of the game. Unlike his first appearance, in which he had a handful of comedic lines, nothing about his appearance in this game is funny.
Gray Mann. In his very first appearance he murders his brothers Redmond and Blutarch in cold blood and takes control of their companies, plots to take over Mann Co., and dispatches an army of robots for that very task that the formerly RED/BLU-employed mercenaries must band together to repel for the sake of their jobs. Now, this kind of diabolical behavior is par for the course in TF2, but what sets Gray apart is his dead seriousness compared to the other characters and the absolute lack of humor in his actions and schemes, in contrast to the kind of stuff usually seen in the game and its side material. Even the premiere video of Mann vs. Machine is noticeably darker and more solemn in reflection of just how much Gray and his robot army are playing for keeps. Though he's not completely serious, considering his robots run on piles of money.
The Classic Team also qualifies, seeing as they're more serious, competent, and organized than their modern counterparts. They don't approve of the antics played around them. Modern Medic has done quite a bit to get on their nerves. Classic Heavy, however, would appear to be the worst of the bunch, as he doesn't hesitate to kill off his employer once he realises the Australium he's after is more valuable to his team than money.
According to Gray's dying words, the Administrator is set to be this, as what she plans to do with the Australium is supposedly worse than anything Gray would do.
Mass Effect 3 has an amusingly literal example in the form of Kai Leng; the Illusive Man's right hand and top Cerberus assassin. When Shepard meets Kai Leng, he's attempting to assassinate the salarian councilor. He (or she, if the player left the council to die in the first game or chose not to import a save) is then killed unless the player has preserved the lives of certain characters throughout the series, in which case he or she is saved by one of two people: Thane Krios or Major Kirrahe. Unfortunately, both characters are critically injured in the process. Kirrahe only gets a few lines out before bleeding out, while Thane gets a final Tear Jerker scene in the Huerta Memorial Hospital. Thankfully, Shepard exacts revenge in the Cerberus base.
The moment Ephidel appears in Fire Emblem 7 is the moment where the game's plot, until then mostly light though with some dark episodes here and there, starts shaping up. The series tend to follow the general "kill some bandits that are anything but threatening and then BOOM the big bad shows up and stuff gets serious", but so far the one guy who managed to turn an entire game from a mostly lighthearted adventure to a dark rollercoaster of death is Manfloy. His actions cause the death of various characters, including Sigurd and most of the 1st Generation playable units, contribute to the subjugation of most of Jugdral to his puppet Arvis' Grannvalian Empire, cause the evil dragon Loptous to posess Arvis' son and start child hunts, and a whole lot of other very unpleasant things for the continent. His granddaughter despises him. The amount of crap this guy did to the mostly peaceful land of Jugdral and its happy map tunes is unprecedented and unchallenged in the series.
Star Fox: Assault has the aparoids. In Star Fox 64, the main villain was a standard megalomaniac, and so was the villain in Star Fox Adventures. The aparoids, however, intend to assimilate all life in the universe. The aparoids destroy most of the Cornerian Fleet, and General Pepper is nearly assimilated by the aparoids, which almost forces Fox to kill him. And Peppy nearly sacrifices himself so Fox and the others can get to the aparoid queen, and Wolf, Leon, and Panther narrowly avoid death as well. They also invaded Sauria/Dinosaur Planet from the previous game, and killed many dinosaurs there including the ThornTails Fox worked so hard to protect before. The aparoids attacked Sauria just to keep Star Fox busy while they launched a full-scale invasion of Corneria, during which they attacked and nearly assimilated General Pepper. They make their entrance by blasting Oikonny, Andross's nephew who comes off as more Affably Evil than anything else. Even the main cast is audibly shocked.
Command & Conquer: Red Alert 2 for the most part is Lighter and Softer sequel compared to its predecessor with most of the characters and plot being based around Rule of Funny, and the threatening Soviet villains are comedic and bordering on harmless. The exception is with Yuri. Departing from the usual hamminess the series Command And Conquer villains usually bring, his behavior is entirely restrained and he talks with deep low voice that coupled with his role involving Psychic Powers outright terrifies a large portion of the fanbase, and the number of moments with him that have any humor to them can be counted on one hand. Come the expansion pack Yuri's Revenge, even with the campaigns maintaining the same humor as the original's, his is still humorless outside of his defeats in the endings and manages to surpass the franchise's Big Bad Kane in his air of menace.
The sequel has the Tyranids in the vanilla campaign. Starting off, it's just tangling with the Orks, which nobody considers a big deal. Then there's the revelation that a Tyranid Hive Fleet is approaching, which proves far more of a threat, and the efforts to stop it are far more desperate.
Lucien becomes one for RuneScape by killing Hazelmere, Cyrisus, and four other heroes. Sliske takes it to an even higher level by assassinating Guthix, starting Gielinor's second God Wars.
Most of Kingdom of Loathing is wacky and silly, but the comedy takes a noticeable downturn when facing certain foes, such as Dad Sea Monkey or the Guy Made of Bees (and in the "Bees Hate You" challenge path, the latter killsthe Naughty Sorceress).
Bugs Bunny & Taz: Time Busters features classic Looney Tunes villain Count Bloodcount as its' Final Boss and the ruler of The Very Definitely Final Dungeon. His presence stops a lot of the silliness in the game and makes the final era a pretty scary place. The other villains can be defeated in relatively comic ways (Elmer is kicked off a cliff, Sam is flattened like a pancake, and Babba is tossed into gongs), but Count has to be genuinely outwitted. Oh, and after you make it through his quiet, scary castle, he goes all out to drain your blood.
Lord Brevon from Freedom Planet is this compared to other characters in the story. Whenever he appears — or is even mentioned — in a cutscene, the game always get much darker and serious.
Doppelganger Arle is an attempt at this in Puyo Puyo~n. While most end bosses in the series tend to fit the comedic tone of each game, Doppelganger Arle's appearance and theme towards the end of the fourth game is much more serious and somber compared to the rest of game. Her motivations (wanting to kill Arle out of pure jealousy) and actions (usingSatan's power to capture Carbuncle and lure Arle into a trap) were also taken more seriously as a result, being a stark contrast from what's a rather light-hearted series for much of its existence.
Satan was also this in the first arcade game. Whenever he appears in cutscenes, he's depicted in a barren wasteland with constant lightning striking, contrasting the typical field the other characters are in. The cutscene before you fight Satan takes it a step further, with ominous music and Satan swooping down from the shadows. Battling him also has a sense of tension, due to the more intense music that plays and the sound effects sounding like explosions. To top it off, throughout the whole game his normally goofy personality is downplayed, becoming The Comically Serious at best, due to Arle accidentally calling himSanta. The second game is the first one that depicts him as the Laughably EvilFinal Boss he's better known as.
The Repliforce from Mega Man X4 effectively kickstarted the themes of how the term "Maverick" can be abused to truly horrific levels (though how much of it was earned due to the self-destructive idiocy of the Repliforce is up for debate), as well as showing a firsthand look at the tensions between Reploids and Humans, both of these themes playing a major role in future X games as well as being the main theme of the Zero and ZX series.
Flowey the Flower in the Neutral and Pacifist routes. Perhaps the most chilling quality about him is that he remembers things you did in previous playthroughs of the game.
You, the player, become this during the Genocide route. And your status as this will stick even if you reset your save file.
Everytime Sans shows his Hidden Depths, suddenly the charming game where you live with these charming monsters suddenly gains several levels of mystery and cynicism.
The Amalgamates mark one of the game's darkest and most nightmarish segments, both in gameplay and in the backstory. They're not that bad in terms of personality and actions, even friendly at times, but their nature of agonized scientific abominations and their creation having a massive effect on turning Alphys into the nervous wreck that she is really kick up the horror and drama.
Jeane's appearance in No More Heroes reveals Travis' Hidden Depths, and his true motive of getting revenge for the death of his family, not to mention her particularly Squick-worthy background and relationship with Travis. She's also the one boss that Travis is unable to defeat without a Big Damn Heroes moment from Shinobu.
Jasper Batt Jr. in No More Heroes 2: Desperate Struggle starts the game's Darker and Edgier tone off by the delivering the head of Travis' friend Bishop through his apartment window. There's also the revelation that all the CE Os of Pizza Butt Travis killed in the first game were all his family members, and he's merely perpetuating the Cycle of Revenge between the two of them. Once Travis actually meets him face-to-face, however, the game actively defies any attempt at serious drama by making him the single most ridiculous boss in both games, to the point that Henry even bails out after his Big Damn Heroes moment because the fight is just getting too silly for him.
Handsome Jack from the Borderlands series. While he's a much funnier example than most, Jack is shown to be a horribly Abusive Parent to his daughter, Angel, manages to kill Helena Pierce, Bloodwing, and Roland and captures/tortures Lilith at one point. His tone of voice also changes to become much colder after Angel's death.
NieR has the scene of a grand wedding between the king of Facade and his bride-to-be Fyra when things go wrong in the blink of an eye via the Shade-wolf Roc and a pack of wolves come and kill the bride and most of the guests. Later turns out to be a case of extreme Disproportionate Retribution. Ouch.
M. Bison from the Street Fighter franchise. Whether it's the comics, film, cartoon, or the games themselves, the consistent thing about Bison is that he is a deadly threat and anytime he shows up, things get very serious.
Yuuki Terumi from the BlazBlue series. Though already pretty dark, Calamity Trigger started off pretty light-hearted, with copious amounts of humor. Once Terumi revealed himself, however, everything gets dead serious. And it continues up until his near demise at Hakumen's hand in Chronophantasma. But when he survived in Central Fiction, he's still dangerous as ever.
Izanami as well. Her appearance at the end of CS makes the plot thicken, but it's not until the end of CP that she starts making big impact, and it's continued throughout CF.
RosenkreuzStilette serves as a clone of and a tribute to the Mega Man franchise, with various nostalgic moments and some amounts of humor. Then once Iris shows her true colors, things go south very quickly. You don't even have to go any further than the beginning of the side game Rosenkreuzstilette Grollschwert to see for yourself as she starts said story off by killing a random priest for the heck of it, and that's saying something.
Spyro the Dragon has its fair shares of loud villains who are evil but still have humour. That cannot be said for The Sorceress and The Sorcerer who are both no nonsense villains who have no regards for others. The Sorceress' plan was to kill 150 baby dragons for a spell to achieve immortality and The Sorcerer's plan was to drain everyone including children of their magic not caring if they're hurt and leaving them in an alternate reality.
Ghostbusters: The Video Game starts out with a lot of the same lighthearted humor of the film series, but things start to get considerably creepy when the Spider Witch turns up in the level where the Sedgewick Hotel is revisited. Like the other Node Guardians, she's murdered a lot of people, but we actually see the bodies of a lot of her victims and witness a reenactment of her leading one of her victims to his death. The lives taken by the previously encountered Node Guardians were at worst briefly mentioned, but actually knowing the full detail of the Spider Witch's crimes has quite a lasting effect on the tone of the game from then on.
Fate/Grand Order isn't a very lighthearted game to begin with (at least in the main story), but when one of the Beasts appear, expect things to go downhill fast. They are leagues more powerful than most Servants, have Nigh-Invulnerability on top of that, and their very existence threatens humanity. Anyone Can Die is in full effect, and expect major sacrifices to be made just to stall them or make them vulnerable (though certainly not easy).