This is based on opinion. Please don't list it on a work's trope example list.
Nightmare Fuel / Darkwing Duck
In his debut episode, Bushroot casually decides to make it look like the scientists who bullied him constantly had "an accident." When Darkwing and Launchpad show up to investigate, Bushroot decides to do the same thing to them. Considering that he's usually pretty meek, seeing Bushroot so calmly resort to murder is jarring; it makes you wonder if his experiment didn't just affect his body...
Sometimes when SHUSH asked Darkwing to take over a case for them they showed him what happened to the guys they had who were looking into it before. Like once Darkwing asked what happened to their last agent and J. Gander replied by taking a can of cat food out of his desk drawer. Although, actually, the cat food was just cat food. The other cat food can in Hooter's desk was the remains of their last agent.
Paddywhack from "The Haunting of Mr. Banana Brain.". Monster Clown, feeds on negative emotions, beak filled with sharp teeth, evil beady eyes, spider-like limbs on a squat body, that deep scary voice... GAH! (when isn't hamming up a scene at least). He's the Darkwing-verse version of It. Issue #7 brings him back. And now..Paddywhack is coming to supper!
After Bushroot sees what he thinks is Darkwing and Posey in a compromising position, he tries to cut Darkwing down with a riding mower; when that doesn't work, he gets out a bazooka.
DarkwarriorDuck. Behind the cartoonish slapstick comedy and the hammy over-the-top performance, and you'll see a man who's become so consumed by his madness that an entire metropolis has to suffer. If the big and scary-looking war machines he uses don't scare you than his new and terrifying appearance, his red eyes and angry-looking face will do it, and if not even that than the fact that he sends people to jail for years for such petty things like jaywalking will do it. And if his entrance wasn't enough to make it clear what something is wrong with his head, then Launchpad will make it clear when he tells his story on how he got fired from the sidekick job because he thought they "should arrest the crooks before giving them the electric chair". And then you'll ask where Negaduck went, or where the other heroes of St. Canard, including Morgana, went, or why the democratic government would allow one man having totalitarian control of a city inside their borders, Joseph Stalin style, without trying to take him out. And it ain't stopping there; when Darkwarrior Duck gets his hands on Quackerjack's time machine, he's planning to use it to go back in time so that he can rewrite the Code of Hammurabi so that even "being cranky in the morning" will be punishable by death. Or go back right at the time when the evolution of landwalking animals is being started, and then delaying it until he'd get "few rules straight". But the moment when his insanity hits the high point is when he aims a missile launcher right at his daughter's face, with a closeness enough to get himself blown up as well if he pulls the trigger, while angrily rants on her past "criminal tendencies". Sure he doesn't pull the trigger but Darkwarrior Duck's Knight Templar personality was so great that it made the Justice Lords look like your friendly neighbourhood patrol police officers. They at least had the mercy enough to just lobotomize the supervillains, and not even Negaduck was this extreme when he found out his own daughter had turned against him.
Then there is the main villain's demise: being hit by the ray and having his head explode, just like the lizard earlier (whose head popped like a balloon). And then, even his ghost gets its head exploded.
Taurus Bulba. Amongst the Rogues Gallery of wacky, comical, over-the-top Saturday morning cartoon villains, we've this ruthless, cunning, murderous, and downright psychopathic crime lord played by Tim Curry. He's the only villain besides Bushroot and Darkwarrior Duck who's confirmed to have committed murder, and his victim was no other than Gosalyn's grandfather. Later on, he attempts to do the same with Gosalyn, both to blackmail Darkwing Duck and For the Evulz, showing that not even children are safe from him. When he's rebuilt by F.O.W.L., he thanks them by destroying their base, declaring himself above them and threatening to kill them after he kills Darkwing Duck. When he seeks out Gosalyn for revenge, she's a complete mental shutdown when meeting him. And while he has some hammy and comical moments, they're way outnumbered by his atrocities, making him the show's local Knight of Cerebus.
The whole episode "Twin Beaks". This episode has Bushroot's "corpse", superficially innocent looking videos with puzzling details in the background, mysterious arc words, a Disney Acid Sequence, and an eerie atmosphere in general.
"Dead Duck": Darkwing dies, visits the afterlife, comes back as a ghost, has to deal with Death hunting him. He wakes up and is overjoyed, thinking it was just a nightmare, and then... it implies it's not just a dream.
Darkwarrior Duck's reappearance in Crisis on Infinite Darkwings is disturbing in a subtle way. In the episode where he appeared, it was implied that Darkwarrior was nothing more than a Bad Future version of Darkwing himself and that he would cease to exist in favor of the real Darkwing once Gosalyn got home. But he's present here again, which means that that wasn't exactly the way it was, and which further means that the horrific alternate St. Canard Gosalyn encountered still exists.
The Muddlefoots becoming tentacle creatures when under the influence of Duckthulhu in issue 10. It is not a pretty sight to see such a (mostly) affable family be transformed into something out of H.P. Lovecraft.