In the opening of potential best episode of all-time, Marge vs. The Monorail, Mr. Burns and Smithers are debating what to do with their excess toxic waste - which they've been illegally dumping all around town for some reason (also kinda weird - how Mr. Burns is PERSONALLY handling something as labor-intensive and shady as dumping toxic waste). When Smithers blithely suggests "the playground," Mr. Burns immediately shoots down that idea...because "all those bald children are arousing suspicion."
Mr. Burns is directly responsible for radiation poisoning for a WHOLE BUNCH OF KIDS. This is likely even beyond the cartoonishly evil acts that would lead Smithers to abandon Mr. Burns due to his descent into "comic book villainy." At least in blocking out the sun, it was clear what Burns' motivations were (money!) - but in actively poisoning (and probably killing) children, when the toxic waste could be dumped literally ANYWHERE ELSE? That's DARK.
And then his chief concern isn't that he's done something horrible - but that people are starting to notice. And so he and Smithers start piling toxic waste in trees at a local park instead, where he's eventually caught (thanks to some Boy Scouts stumbling onto his little game of "hide the ooze") and forced to pay a fine. FOR MURDERING CHILDREN.
Although, honestly it is kind of endearing how bad he and Smithers are at hiding their barrels of toxic waste.
No Simpsons character suffers to quite the degree of Hans Moleman - acting very much like South Park's Kenny, Moleman has repeatedly died and mysteriously returned to life as though nothing happened. But his fate in Team Homer puts pretty much every other terrible fate (being brainwashed into thinking he's Bart, forced off the road and dying in a fiery car crash, apparently being physically ruined due to alcoholism, etc.) - hearing that Mr. Burns was being particularly generous (due to inhaling a whole bunch of ether), he made the humble request to have his push-broom rebristled for $17. And instead, Mr. Burns imagined him as Lucky, the Lucky Charms leprechaun....and, for some reason, drilled into his brain.
It's such a dark, gruesome turn for the scene, all the camera can do is cut to right outside the office so we can hear Moleman feebly cry "My brains..." His sad exclamation is somehow even more of a downer than him being put to death...AFTER Homer ate his last meal.
Hans Moleman has had it rough, but asking for a broom improvement and getting unexpectedly drilled through the brain (and probably killed) is one of the most upsetting things The Simpsons has ever done.
You might look at this and think it isn't that dark - after all, nothing happened! No one was injured at all - and certainly worse things have happened to children on The Simpsons than ALMOST getting punched in the face. The key thing here is the intent, though - Krusty the Klown, a character we're supposed to have sympathy for (since the episode is ABOUT him and and his downfall) is preparing to PUNCH A CHILD IN THE FACE OUT OF SPITE.
And not just ANY Simpsons punch - look at the detail the animators gave Krusty's raised and shaking fist - each knuckle is detailed, the fist is drawn more realistically than Simpsons' character hands ever are, and there is pure rage in Krusty's face. He is so angry, he wants to brutalize Bart's face. Krusty's been low before, but this is almost definitely his lowest - and darkest - moment.
The Simpsons wanted to riff on the Hollywood sex scandals of the mid-90s - particularly Eddie Murphy and Hugh Grant - through the lens of resident washed-up actor, Troy McClure. But in an effort to take things a bit further and more exaggerated for the wacky world of Springfield, the writers decided to up the ante and give Troy a fetish that is...well, a whole lot more disturbing than maybe any other sexual fetish I've ever seen on television. In short, it's heavily HEAVILY implied Troy McClure has sex with fish.
I'm honestly not even sure HOW someone would go about trying to have sex with a fish - and that's what makes the whole thing so, so dark. Given you'd also have a hard time getting consent from a fish, the only possible conclusion is that Troy rapes fish. I don't know precisely what he does to them, but he is sexually assaulting fish - and even HE knows how dark that is:
And in case the implication wasn't already crystal clear, this piece of dialogue (that I believe was cut from syndication) between Fat Tony and Louie at the DMV makes it even more explicit: