The Archmage in Grim Grimoire. 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 and Dangerously Genre Savvy, 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.
Kurtis looked like one of these in the firstDisgaea 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)!
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 Games are a barrel of laughs and fun to play, although 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.
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.
Final Fantasy V brought us 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 also this to the series in general; with the exception of Cloud of Darkness, all prior villains were generic Tin Tyrants who wanted to take over or destroy the world, and aside from being Evil Overlords none of them got much characterization. Kefka on the other hand quite clearly loved the carnage and destruction he was causing and gleefully Kicked The Dog every chance he got just for the hell of doing it. Since him, the villains of the series have become more sinister and ambitious.
Mortal Kombat 9, as per its series tradition, is normally gory, but things do start out looking good for the heroes despite Raiden's attempt to change the timeline. Then Shao Kahn becomes one by killing Kung Lao right after his victory over Kintaro, showing that Raiden's actions have started to have disastrous effects. The bad news, that's just a minor one. The next time, Sindel becomes empowered with Shang Tsung's souls, and murders the majority of the good guys, effectively putting Raiden and the Forces of Good at a severe disadvantage.
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. 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 in an And I Must Scream situation, 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.
Mega Man Zero was already the darkest series in the franchise to begin with, but when Dr. Weil appeared in the third game, the already dark series got even darker, making every effort he can make life a living hell for humans and machines alike.
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.
Trials and Tribulations: An odd case here in Dahlia Hawthorne. The villainess of three of the games 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.
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 BSOD 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 pretty much 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.
The game was not necessarily all bubblegum and rainbows, but Ryoji's reveal as the Avatar of Nyx marked a dark turn in the game's story, along with the Moral Dilemma to either kill Ryoji, thus erasing your memories of the apocalypse to come and allowing you and your friends to live in blissful ignorance until the end; or spare Ryoji, thus allowing him to fully become the Nyx Avatar, and possibly dooming yourself and your friends to a long, excruciating end.
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.
Shadow the Hedgehog's introduction into the Sonic the Hedgehog 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.
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 robot (not in the usual "stuff him into a Badnik" way, but SatAM's "turn organism into robot" method), but due to a rescue by Tails, they capture him instead, but try to turn him into a robot anyway. Eventually, Amy and Knuckles get their lives drained by the machine, and later on they (seemingly) kill Eggman. Oh, and Zavok taunts Sonic about all this throughout Sky Road, to the hedgehog's anger.
The Kingdom Hearts series has always been lighthearted even when being grim and morally deep, being a Disney crossover after all. However, the arrival of Master Xehanort in Birth By Sleep made the game the darkest game of the series, depicting Eraqus being beaten down by Terra before Xehanort finishes him off, Master Xehanort possessing Terra's body to become the current Xehanort, Ven being sent into a coma when he destroys his own heart, and Aqua being trapped in the Realm of Darkness. Heck, the Sequel Hook for the game at the end of Kingdom Hearts II serves to show this guy is in a league of his own, showing him freezing Ven solid and raising mountains of earth with a flick of his hand, instantly making him the strongest being the series had seen up to that point.
In general, Xehanort or any of his incarnations are this. Part of Birth By Sleep was darker than the other games was because Xenahort had more screen time and a direct and active role in than any early game in the series.
Ghetsis from Pokémon Black and White. Would you like some horrific 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 and a damn sight nastier than just about anything that has come since. 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.
As proof, the fanfic Ashes to Ashes pits May and Maxie against Cipher - Maxie already has somewhat sympathetic motives in the source work, so naturally compared to Cipher, he's an absolute saint.
Purple Eyes from Pokémon Ranger Guardian Signs fits this trope to a T. Let's see, before we even meet him in person for the first time, he's beaten Rand badly and kidnapped his wife and daughter, ouright 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. He's certainly unique as Pokémon villains go, and is pretty much Pokémon's answer to Kefka. 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.
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.
Despite the games being seemingly lighthearted, most villains in Kirby can qualify for this. Dark Matter, the Nightmare, Zero, Magolor's Soul, Landia, etc.
Chaos Lord Ledgermayne of AdventureQuest Worlds counts as well. 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, also 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.
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, but whenever Ghirahim appears, the game makes a big change in tone, becoming much more serious. His owner, Demise, isn't much better.
The Flood's appearance in Halo: Combat Evolved could count. Granted, it was a pretty serious story to begin with, what with aliens declaring humans heretics and attempting to purge us from the galaxy, but it was a pretty standard sci-fi alien-shooter story, and the enemies weren't too threatening (the Grunts especially were too whacky to take seriously) . Then we get to 343 Guilty Spark and have to deal with those gurgling abomninations that just won't stay dead. Along with the threat of them getting loose and finding earth, the story went from "resist the aliens" to "save the galaxy" pretty quick.
While Soul Nomad & the World Eaters never where really bright and happy to begin with things turn viciously sinister when the Devourlord enters the stage.
Gray Mann in Team Fortress 2. 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 we usually see 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 the new gray team is 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.
Mass Effect is another series that wasn't exactly light and happy from the beginning, but the series takes a dramatic turn for the darker on the planet Virmire. Things get bad from the beginning, but then Commander Shepard meets Sovereign, whose appearance heralds the apocalyptic mood of the rest of the series. The Collectors and by extension Harbinger then one-up Sovereign in the opening scene of the second game, destroying the Normandy and killing Shepard. The heroes never even get a chance to fight back before getting utterly defeated.
The third entry gives us 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.
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.
Command & ConquerRed 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 Ptolemaic Army in Metal Slug 5 and the Venusians in Metal Slug 6.
Dawn of War, despite the Warhammer40000's reputation for grimdarkness, starts off as fairly typical war plot. Then Sindri appears, bringing up the threat of the Maledictim and manipulates Isador into Face-Heel Turn. The expansions prove less serious thanks to a general lack of any villain that a presence he did.
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.