In Blood Bank, Jack the Curator's arrival signals that the story is about to get much darker.
Bravoman: In the webcomic, Bravomaster is a heroic example, while his enemy, Zulu, still get's some funny moments from beating up the Bravo corps. they combine into Bravomaster and toss him into the sun, the lack of jokes on panel is later lampshaded. Later, Bravoman get's transported to an alternate universe where the world has been destroyed. Zulu had come to Earth and forced everyone to team up to stop it, having done so, Dr. Bomb unleashed his army on the heroes, shutting down the fourth wall and preventing any gag-based powers or easy victories. Earth has been reduced to a wasteland and Dr. Bomb was deposed long ago, and it was really the version of Anti-Bravoman who had done all of this, from unleashing Zulu, to shutting down the fourth wall even to attacking Salaryman and imprisoning him. It was all in the quest to become stronger, and in the end the heroes have to return the meta-humor to the strip to stop him.
But I'm a Cat Person: It's around the time that we learn more about Ann Walker's goals that the mood of the comic takes a sharp turn from a fairly light-hearted fantasy comedy to a dark urban fantasy adventure.
The second master, Noisemaster (alongside the third, his cohort Mutemaster), is much more cunning than he lets on (particularly compared to Splashmaster, the first boss), and nearlywipes out an entire city via Kill Sat.
The Dragon Doctors: The originally only somewhat-serious strip gets a lot more heavy once the Crax chapter begins. It's a horrible flesh-and-mind-devouring parasite, and it's followed up on with a serial killer who kills people with nightmarish death spirits, a Die Hard scenario in a hospital, and tragic backstory after tragic backstory.
The first two of the Four Fiends, Lich and Kary, are played as fairly serious villains, with their presence resulting in major character deaths (and in the case of Black Belt, Killed Off for Real) and some dramatic moments. The later two, Ur and Muffin subvert this trope, with Ur being killed in an Anticlimax while the Muffin Arc is dealt with humorously as usual.
And then we get Black Mage's "Almighty wizard of infinite evil"-form, which, unlike all other "evils" up to that point, actually causes a major Cerebus Syndrome and manages to kill off half of the cast in less than 10 comic strips before squaring off for the final battle with a powerful wizard. Until Sarda comes in and reveals his plan, that is.
Damien is a great example of this trope, to the point where the author killed him just to stave off Cerebus Syndrome. It didn't really work, as the series has since entered Cerebus Syndrome from a different angle.
Abraham was supposed to be even worse, but the whole thing turned out to be one huge misunderstanding on his part, and after everything was explained to him, he surrendered without further fight. He even got to be funny in places, something that did not apply to Damien. Pandora seems primed to do this in-universe, as she seeks to give her son and godson a world where they can make a difference.
Fifteen Minds: The appearance of the fortune teller on page 9 of 31 in Blue Moon Blossom is when the plot suddenly kicks into gear, and shifts the story from episodic Scenery Porn to a straight-up adventure, albeit still a very beautiful, Scenery Porn-laden one.
Freefall gives us Mr. Kornada, initially an incompetent and seemingly unaware low-level executive with Ecosystems Unlimited. As the plot unfolds, we learn that he has used his position (toothless as it is) to commandeer his high-ranking nephew's robot assistant Clippy for a scheme to make himself the wealthiest man on the planet at any cost. In the process, he inadvertently opens up a can of worms regarding civil rights for AIs, which drives the bulk of the first book of the comic.
Homestuck: Given the ever-escalating nature of the story, there are at least three "candidates": the meteor at the end of Act 1 (not a character, but the moment where things start to shift from inventory shenanigans to plot), Jack Noir in Act 4 and Lord English, or alternatively his servant Doc Scratch in Hivebent/Act 5.
The Midnight Crew intermission also has one with Snowman. While the Felt were otherwise portrayed as hilariously incompetent gangsters existing only to mess up with their time powers before getting killed by the Midnight Crew, Snowman came in, and spent the rest of her screentime horribly maiming the fan-favourite main character.
Before Lord English's appearance, every major villain had some sort of comedy to them. Bec Noir wanting bones like a dog would, Doc Scratch as serious as he usually is has some humorous lines. Lord English has yet to do anything supposed to be perceived as comedic. In fact, the first thing he does after his introduction is to go and kill the author, who was absolutely terrified when he appeared. He then proceeded to presumably erased many dead characters' souls from existence with a single mouth laser. Although, his younger self is quite a humorous villain.
The Inexplicable Adventures of Bob!: Coney and Gosh serve this role. As Bob and Jean discuss at the end of the Cone arc, Bob comes close to getting killed all the time, but his experience inside the Cone Ship is the first time it's seriously, lastingly scared him. The story still ends on an upbeat note, but Bob seems much more aware of the precariousness of his situation than he's been up to this point.
Ozimaar appeared unexpectedly on page 14 after 13 silly comics and created a nonsensical storyline which lasted 'till December '07 when the author cancelled the arc and skipped on ahead in the story, because the Ozimaar Arc was boring him.
Then Computer re-appeared rampaging through the ongoing story lines and twisting them onto her self.
Kiwi Blitz features the villainess Gear, whose presense is currently the only source of grimness and darkness in an otherwise fun and whimsical webcomic.
Last Res0rt was already pretty dark, but by the time we bring Veled around... she's the Big Bad, and she doesn't leave much doubt of it either. Even if she does have more comedic value than most as she's easily bored (and eager for entertainment), Veled never comes off as harmless — in fact, when she's bored, she's even more dangerous.
Modest Medusa: The chainsaw unicorn starts a rather bloody fight in what had previously been a lighthearted comic with no action and heralds the start of the Story Arc.
Nale marks the beginning of the comic's first plot, and while he's very much still a comedic character, he has the honour of causing the first wholly serious moment in the comic by literally stabbing Elan in the back.
Miko is responsible for introducing the cast to the overarching plot by arresting them for damaging the fabric of reality. Quite what she means by this isn't immediately obvious, but when it becomes clear, the story kicks fully into action and enters its Myth Arc proper.
RPG World: Jeff kills Eikre's family and makes some pretty disturbing scenes, giving a whole lot of drama to a webcomic parodying classical RPG cliches.
Sam & Fuzzy takes a turn for the serious which is arguably kicked off by Fridge. Although he starts off as comic relief, his actions in the "Friday Night" arc drag Sam into the Ninja Mafia plotline that has dominated the last four years of the comic.
Schlock Mercenary has played with this trope before, but it didn't stick until the introduction of Admiral Emm. The only reason the Toughs weren't immediately compressed into neutronium and fed into an annie plant was because Admiral Emm wanted to let Colonel DeHaans torture them first, just to make sure her clean-up job on Laz'R'Us nanite information was complete and thorough. The Toughs only survived by handing over an expert on immortality treatment and agreeing to let the UNS mindwipe them. The fact that the very next story arc was the darkest story arc by far didn't help anything.
Scoob and Shag: The comic starts as a series of unconnected gag-a-day strips, focusing on light comedy and goofiness and with no overmatching plot. "Mick"'s appearance in a Haunted House strip marks the comic's turn into hard drama, introducing strong horror elements and starting the comic into a full Myth Arc.
Sinfest: Xanthe brings in the Sisterhood arc after which much of the series' humor has vanished. An interesting example in that she doesn't seem to be a villain, though her extremist views tend to annoy the fans anyway.
Sleepless Domain: The Elite Mook from the second chapter. At first glance it's just a creepy monster. Then it kills three members of Team Alchemist and leaves a fourth, Undine, at death's door. Undine survives only because the team's leader, Tessa, heals her at the cost of her own powers.
Undine: On that night... before any of us knew what was happening... our team went from five members... to one.
The strip kicks its Cerebus Syndrome off with a Mook vampire named Kullan. He fits this trope because his introduction is (a) pretty much the first strip in the series to end on an ominous note rather than a straight-up funny one, and (b) the start of the "Vampires" arc, Sluggy's first more or less serious storyline.
As the strip progresses, the presence of Oasis, K'Z'K or HeretiCorp is a fairly good signal that a given arc is going to be darker than usual.
Dr. Schlock may qualify. Not only is he involved in virtually every other arc's sinister events, but even his first, still-comedic appearance, as a scientist testing cosmetics on Bun-bun (or, well, trying to), sets up numerous events in the series, including the nanomachine plague. It's not clear if that cosmetic lab (which also did nanomachine research) was part of HeretiCorp or not (and, if it was, if it had been all along, or if Abrams decided retroactively that it was).
Spinnerette: Colonel Glass. Before his appearance, all the series had for major antagonists were Dr. Universe, Alexis Woodrow, and Captain Alberta — a noble Well-Intentioned Extremist, a selfish Attention Whore, and an incompetent Politically Incorrect Villain respectively. Glass is an out-and-out Ax-Crazy sadist who kills and skins animals to send messages — a modus operandi disturbingly similar to that of the notorious House Bolton. Glass is also notable for being far more powerful than most supers in the Spinnyverse. In a setting in which most characters have abilities like moderate super-strength, a weaker and more realistic version of the Spider-Man power set, or sonic punches, Glass is essentially Magneto with Wolverine's Healing Factor: he can control glass with enough power to chop up people and vehicles in seconds and create near-impenetrable shields, and enough finesse to grab hostile drones in midair and send them crashing into their controllers, not to mention regrowing lost limbs and shrugging off whole-body third degree burns. His appearance pushed the comic permanently to the Darker and Edgier side of spectrum: the Wide-Eyed Idealist Spinnerette has to resort to killing him with thermite, and Mecha Maid is traumatized.
Stand Still, Stay Silent: Fighting Plague Zombie monsters was given considering the crew's mission, but their early opponents were whatever happened to be living in the location they were visiting, and obeying to their violent instinct. Then a ghost with murderous intent assembles a mixed herd of trolls and other ghosts whose sole purpose is to follow the crew and kill them off. The first battle between that herd and the crew resulted in major damage on their transportation, left the most powerful mage able to help them in a Power-Strain Blackout-induced Deep Sleep that lasts for several days and, after some delay, the death of one of the non-immune members of the crew. The humor doesn't completely disappear after the events, but has become more prone to being Black Comedy.
Zebra Girl has always kept a somewhat uneasy balance between funny and dramatic, but it tilted firmly into dramatic territory and ditched some of its sillier, Fourth Wall-breaking gags with the appearance of Harold DuVase. This is kind of odd, since DuVase appears at first to be a Harry Potter parody. In the end, he turns out to be something much, much worse...