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Suburban Knights has their Big Bad, Malachite. He scares the living crap out of the regular cast, and with good reason. Even the one joke in the whole crossover his involved just serves to to add to how horrible he is.
The sequel follows suit with the following Big BadThe Executor, who while not as serious as Malachite, is a lot more dangerous since he plans to wipe out Earth and any world that opposes his rule.
Mechakara also counts for this one, maybe even more so than The Executor. He is the only villain in the entire feature who is never played for laughs and even Zodd and Turrell are terrified of him in the end. Word of God says that Linkara was very protective of the character, and wouldn't let Doug write anything that would cause Mechakara's threat to be Played for Laughs. They were even originally planning on doing the switch for Suburban Knights, with Mechakara being forced to play along with the antics, but Lewis argued that Mechakara would rip everyone's throats out before participating in a fantasy quest, leading to his role in To Boldly Flee.
Demo Reel was a pretty sad show anyway, but it got sincerely depressing whenever Donnie's mom got mentioned. For good reason too, as she was an aging actress who committed suicide, leaving her only child alone with nobody who gave a damn about him.
Reflets d'Acide starts out as a light-hearted Heroic Fantasy parody with no villains beside monsters met by the heroes. Then, we get:
Alia-Aenor, who epicly double-subverts this trope; when first seen, she appears as an intelligent, humongous black dragon. A few minutes later, she turns out to be a female, whose human form, much to the narrator's dismay, is a beautiful girl with a sweet, girly attitude... then she kills a bunch of thieves trying to rob her with a single spell.
Bélial, on the other hand, plays this trope straight.
Geronimon in Godzilla and His Amazing Friends. While previous enemy monsters weren't exactly lighthearted, he's the first one to be depicted as explicitly out to kill the group and have planned to do so (previous monsters were primarily predators or space monsters the group happened to come in conflict with). Word of God has stated that Geronimon's goal is to kill Godzilla's friends one by one, then kill Godzilla himself, and that he's done something in the past that Godzilla will never forgive. He also has the dangerous ability to resurrect other monsters. Not only do he and his monster army nearly beat Gomora and Godzilla to death, it takes the entirety of Monster Island's inhabitants to stop them. And on top of that, he manages to brutally kill Gomora, a recurring ally, by stabbing him to death with his feathers and escape before Godzilla can take his revenge.
The Ask Princess Molestia blog is typically about the wacky and lighthearted hijinks of a Memetic Molester and an AdorkableGamer Chick. However, whenever Fausticorn swings by for a visit, it careens into a rather dark and depressing drama that shows Molestia has some seriousissues. Give it a few posts and it's back to normal, but after that first visit, things never quite made it back to the original silliness.
Similar to Atop the Fourth Wall above, The Spoony Experiment has managed to pull this trope off. The first was with Black Lantern Spoony, who tries to kill the clone Spoony and take the show over. Unlike Spoony's other would be murderers who are dealt with in comedic ways, there is nothing humorous about him. His current arc has Sephiroth. While his exact goals are unknown, he has no comedic traits to him, and the hints we have of his plans are pure Nightmare Fuel. The Guardian, the Big Bad of the Ultima story arc, in fact states he had no plans of conquering Earth because Sephiroth's (though he doesn't refer to him by name) Evil Plan, he feels has already doomed the planet.
Red vs. Blue started as a more comedic series than anything, but towards Out of Mind and Recovery One, it took a turn into dramedy territory, all thanks to the characters below.
Recovery One onwards gives us both The Meta and Agent Washington. The former is an Ax-CrazyGenius Bruisercontrolled by a psychopathic AI unit that is first introduced by wiping out several members of a Redshirt Army (in the process making the Laughably Evil Omega a Not-So-Harmless Villain), the latter is a ruthless Anti-Hero that will willingly turn on someone without remorse if they betray him. The pair caused the series to dip into dramedy territory, rather than straight up comedy. Wash has a villainous stint from the end of Recreation up till the end of Recollection in which he and Meta team up, causing the show to dip into drama again.
Oh but Locus is nothing compared to his partner Felix, who reveals that not only have the two been playing both sides of the civil war to wipe out Chorus' entire population, but that he used the heroes to help him perpetuate the hate the two sides have for each other. Unlike Locus, Felix is completely sociopathic and enjoys twisting the knife whenever he feels it would be most devastating, seemingly taking joy from genocide. What makes him stand out is the fact that before The Reveal, he was funny, but then immediately dropped all pretense of humor afterwards.
Epic Rap Battles of History is generally a lighthearted-ish show, though it has a couple of odd examples, and a few played completely straight.
Stalin appears in the Season 2 finale. Unlike most examples of this trope, he's still played for laughs, but the Black Comedy is taken Up to Eleven. Furthermore, some of the stuff he says comes across as less amusing and more terrifying in now small part owing as to how casually it's said, and quite a few comments directed his way are genuine revulsion rather than petty jabs. He also causes Lenin to come back from the dead and rant (well, rant through rapping) at him for his tyranny. In short, compared to such real life maniacs like Adolf Hitler and Genghis Khan, both of whom are Laughably Evil in the show, Stalin is downright sinister.
The same episode inverts it with Mikhail Gorbachev. Before he showed up, things had been reaching really dark territory. When he shows up again, it gets significantly lighter.
Season 3's Scrooge vs Donald Trump brings us The Ghost of Christmas Yet to Come. While he's not 100% evil, he isn't even played for Black Comedy laughs, has a terrifying design and voice and ultimately scares Scrooge into a Heel-Face Turn by pointing out how he will die alone and unloved. This version also takes the Grim Reaper aspects up a notch, having him appear as a withered skeleton. His lyrics are terrifying, too, and he enters with an In-UniverseJump Scare.
In Season 4, the battle between Jack the Ripper and Hannibal Lecter is notably darker than the usual rap battle. While there still the occasional funny line, both rappers are incredibly serious and unabashedly blunt about the brutal horrific murders they commit. The entire video is the closest ERB gets to Nightmare Fuel from the intense, unnerving beat to the Jump Scare at the end.
From This Is It's video Don't Hug Me I'm Scared II: TIME, we are introduced to an anthropomorphic clock named Tony. Throughout the video, he attempted to teach the other puppets that there was enough time in the world for them to do everything they wanted in life, including watching their television show. However, as the journey and song progressed, Tony became more and more controlling and forced the puppets to act strictly on his schedule, and then began to buzz loudly in front of them when the puppets began to show signs of resistance to his teachings about time by wandering about it being real, or a human perception. He was beeping so loudly, that he caused Manny's ears to bleed. He then used time to cause the puppets to age rapidly and fall apart in bloodily ways. He then ends the song by stating that everyone eventually runs out of time. He's also shown to be more outright vile than the Notepad from the first video.
Cell in Dragon Ball Z Abridged seems to be this. He hasn't even appeared in person yet, but already he's caused the tone to shift from humorous to serious in Episode 40. No villain prior, not even Freeza, has been treated this seriously, and said tone shift is lampshaded by (a hopped up on LSD) Mr. Popo:
Mr. Popo: Well that's f[beep]ing ominous.
His first real appearance only cements this. He appears dragging what might be a (wet) corpse around through a shadowy back alley, nonchalantly stalking Piccolo while singing "Mr. Sandman" in a voice that sounds like it belongs to a sixty year-old chain smoker who just finished his fifth pack of the day. This is after he killed off a whole city and absorbed them.
Cell: Mr. Sandman...bring me a dream...make him the cutest that I've ever seen...Give him two lips...like roses and clover...Then tell him that his lonesome nights are...over.
Zig-Zagged and Lampshaded when he gets actual screentime though. He's the darkest and most disturbing villain of the series thus far, and his introduction, as noted above, is played as chillingly as possible, but he's full of Black Comedy, almost as if he's aware of his status as a Knight of Cerebus and is having fun with it.
However, his Knight of Cerebus role is overshadowed by Wilford Brimley, who traumatizes Madotsuki and manipulates the other members of Los no Frollos (Batiatus included) to gain limitless power for himself. He succeeds, as he absorbs Madotsuki's sorrows and takes Panty hostage to create an army of perfect soldiers.
The Lizzie Bennet Diaries starts off as mostly a regular rom-com, with short flashes into more serious things such as the Bennet family's finances and the disputes between Lizzie and both Charlotte and Lydia. The story gets exponentially darker when George Wickham enters their lives, ends up dating Lydia, is psychologically abusive and manipulative, almost publishes a sex tape of himself and Lydia for money without her permission, and denies the entire thing later on when Gigi, Lizzie's friend (and sister of Lizzie's love interest Darcy), confronts him about it. (Keep in mind that Gigi dated Wickham once herself, and it ultimately ended up that he was only interested in her because of her money.) He seems to completely get away with it, even though the tape is never released thanks to Darcy's intervention. The worst part about it is that it is heavily implied that Wickham did it just to get back at Lizzie for siding with Darcy instead of him over a dispute between the men that was ages ago and didn't even involve her.