- Nomad of Nowhere:
- Episode five introduces us to The Undertaker. Before then, all the villains after the Nomad have either been incompetent (Red Manuel), silly (Don Paragon), or decent people that just happened to be loyal to bad people (Captain Toth and Skout), but The Undertaker is just a completely creepy and sociopathic grave digger with a rather morbid sense of humor, who puts on phony friendly demeanor to abduct and imprison the Nomad, so he can satisfy his his scientific curiosities about dark magic. During the episode he kills/destroys two of the objects the Nomad brought to life and at one point even tries to dissect the Nomad alive. Tellingly, after he destroys the second object the Nomad brought to life, the normally pacifistic Nomad comes close to killing The Undertaker before he manages stop himself at the last moment.
- In the very next episode, we finally get a glimpse at the series' Greater-Scope Villain, King El Rey, when the Nomad starts getting flashes of the kings past and we learn that El Rey used to be The Good King who used his magical crown to bestow gifts onto his subjects. But when his crown started to run out of power, he refueled it by devouring magical beings and became increasingly tyrannical as time went on, revealing that he is the reason The Nomad is the last known magical being in the world.
- While Red vs. Blue has always been an overall comedic series, as the story progresses, characters are introduced that would make the series much darker in tone, turning it from a relatively plot-light Work Com to a much more plot-heavy dramedy.
- Wyoming in The Blood Gulch Chronicles is a downplayed example. While he's still as Laughably Evil as Omega, he's treated as a much bigger threat as well as the first truly dangerous villain that the Reds and Blues face.
- The Recovery One miniseries introduces both Agent Washington and the Meta. The former is a ruthless Anti-Hero that will willingly turn on someone without remorse if they betray him, while the latter is an Ax-Crazy Genius Bruiser controlled by a psychopathic A.I. unit that is properly introduced by wiping out several members of a Redshirt Army (in the process making the Laughably Evil Omega a Not-So-Harmless Villain). The pair's introduction marks the series turning point into a dramedy rather than the straight up comedy it had been up to that point.
- The Recollection introduces two characters whose appearances would establish the much heavier focus on plot for the later seasons:
- The Director, by virtue of being the Greater-Scope Villain of the entire series up to that point, is one of the darkest characters in the series, with his back and forth messages between him and the Chairman establishing the plot-heavy focus the series would be taking from then on.
- The Counselor's introduction scene is the first scene in the series to be completely devoid of humor, with his implied order to execute a survivor of the Meta's rampage establishing the how much darker the villains of the series will be compared to the Laughably Evil villains seen previously.
- Omega, of all characters, becomes this upon being reintroduced in The Project Freelancer Saga. Despite his role as a Laughably Evil Harmless Villain in The Blood Gulch Chronicles, the moment he appears in any scene of this arc, all humor goes out the window.
- The Chorus Trilogy:
- Locus, a One-Man Army mercenary whose first appearance causes the arc, which up to this point had been more similar in tone to The Blood Gulch Chronicles, to swing right back into the dark tone of the other story arcs. It also helps that, while he has his moments of comedy as The Comically Serious, he's almost never Played for Laughs.
- Fellow mercenary Felix also acts as this, with his appearance establishing the plot of the arc. And then it's revealed that he was Evil All Along, and things get even darker.
- While the Merc prequel episodes of Season 14 were already some of the darker episodes of the season, the appearance of Ruben Lozano, a crime boss Felix, Locus, and Siris have kidnapped the son of, causes the episodes to become even darker.
- While his actions result in a lot of comedic moments, any time Labyrinth physically appears during Singularity, he's played completely seriously.
- The Frollo Show has Wilford Brimley who overshadows every previous villain in the show in threat level, 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 Yogscast Minecraft Series tends to be mostly amusing, focusing on the Yogscast and their somewhat bizarre antics in Minecraft, but throughout the years there have always been some darker villains that cause the humour level to drop:
- Shadow of Israphel primarily has the titular antagonist, who changed Lewis Brindley and Simon Lane's lighthearted Let's Play into a somewhat more serious show. Initially just a player that harassed them and caused the two frustration, his more horrifying traits have been highlighted more, with his funny ones being phased out of anything canon. He was also revealed to have murdered Knight_Peculier's mother in an outright terrifying flashback, also turning KP's life into something hellish and nearly driving him to suicide.
- Feed the World was a series revolving around Sjin building a farm and a compound, but episode 25 introduced Strawfingers, a scarecrow that kidnapped Kim Richards, took her to the Nether, fed her chicken and stole Sjin's textures. This was initially a somewhat humorous setup, but later reveals about Strawfingers, namely his murdering Abel's parents and planning to nuke the Sjindig, rendered him in a much darker light.
- Yoglabs has a downplayed example with Gozencratz and Rildernstern. Compared to all the above examples, they are downright comical, but Yoglabs typically stuck with a Lighter and Softer theme, with most episodes revolving around a minor accident. The duo planned to Take Over the World and their actions have long-lasting consequences that impact the next series, with the implication that something worse is inbound. On a more worrying note, Israphel briefly appears during an episode, and while not there for long the episode loses all humour when he's onscreen.
- Llamas with Hats: The show switches from being a surreal Black Comedy about a Laughably Evil mass murderer to a Dramedy about a legitimately disturbing (and disturbed) Omnicidal Maniac right around the time the Paul Mask shows up.
- MyStreet was originally a Lighter and Softer counterpart to Minecraft Diaries, transplanting many of that series' characters into a comedic Slice of Life setting. Then came Emerald Secret and the arrival of Ein, The Sociopath and a Hate Sink who, among other things, brainwashes Aphmau into becoming his lover and trying to kill Aaron. This ended up marking the series' permanent shift from lighthearted comedy to a darker fantasy-drama.
- Puffin Forest tells stories about his Tabletop RPG misadventures with a strong emphasis on comedy. That means even when he talks about canonically vile antagonists or those that win, it is often in a comedic context, be it by the party's actions, DM mistakes, or just whims of the dice. That changes once he starts doing a longer series about a particular module and it's villain, Strahd Von Zarovich, quickly puts the entire party through a series of nightmarish scenarios and tragedies while playing them like a fiddle and getting much more depth than usual antagonists.
Knight Of Cerebus / Web Animation