In Adventure Time, the first episode of the sixth season's climax ends with the death of the minor recurring character, Prismo.
A few episodes later, an even more minor character (Root Beer Guy) ends up dying in an Heroic Sacrifice. Even after everyone else that died in the same battle he did ends up being revived, he stays dead. The episode is then dedicated in his memory.
In a subversion of the very trope named after him, the Joker eventually got this treatment in the DCAU. Yes, having proven himself as much of a survivor as his counterpart in the comics, every bit the "no one" in No One Could Survive That, Batman Beyond: Return of the Joker took the big leap and put him down once and for all - killing him twice; first his body was destroyed and buried deep within Arkham Asylum in flashback, then the chip containing a copy of his DNA and memories was burned out, just to make sure he won't be coming back. (In just a bit of a cheat, the event occurs at the far end of the universe-at-large's history, allowing him to show up in stories taking place earlier; Justice League took advantage of this.)
Malware on Ben 10: Omniverse is permanently done away with in the episode "Showdown Part 2" when Ben regains Feedback after a talk with his past self, then Azmuth helps to increase his power to take Malware down.
The Boondocks Season 1: "Granddad's Fight." The karmic fate of life-long, Five-Star Magnificent Bastard Colonel H. Stinkmeaner, who effectively gets his ass handed to him by Robert Freeman in an ironic Curb-Stomp Battle: Stinkmeaner had beaten up Freeman in an argument over a parking spot, and the entire episode revolves around Huey making him out to be a version of a blind assassin, and Robert training for a rematch. It turns out Stinkmeaner was just a crotchety old man who got lucky. Unfortunately, he's such a temporal Jerk Ass, absolutely nobody realizes this until Robert pulverizes him. The beating is so bad, Stinkmeaner ends up stone-cold dead with a solid punch to the face. Feeling remorseful at his grave, Robert prays to God for forgiveness the end of the episode.
Although dead, Stinkmeaner makes one hell of a return in Season 2: "Stinkmeaner Strikes Back"- literally. Stinkmeaner has been sent to hell after death, but now proves he can seriously fight back- by pummeling his way through every demon who steps up to challenge him. Satan himself is so marveled at Stinkmeaner's badass exposition, he sends him back to Earth to spread ignorance and chaos among the black community, also giving him the opportunity to seek revenge against the Freeman family. As a ghost, he posesses Tom Dubois and trashes most of his house. After Uncle Ruckus and Riley fail miserably at exorcising Stinkmeaner, Ghostface Killah's spirit shows up and clues Huey in to the solution: put Stinkmeaner at peace by having Ruckus spout off about how he hates black people, a hate Stinkmeaner firmly shares with him. Moments later, he's exorcised and sent right back to hell.
In yet another Stinkmeaner-related episode, death strikes again, this time nailing Bushido Brown, via on-screen decapitation during his fight in Season 3: Stinkmeaner 3: The Hateocracy". The Colonel apparently palled around with a group of disgruntled elderly, nigh-crazy jerkasses who formed the Hateocracy, a gang that wreaked havoc within the walls of a nursing home. The members of the gang show up to curb stomp the Freeman family in what looks like a Roaring Rampage of Revenge, so the Freemans hire Bushido as their equally jerkass bodyguard, who actually holds off the Hateocracy- until he catches a spinning disk of doom to the neck. The Freeman family's reaction? A group "OH, SHIIIIT!".
It gets more bizarre when Stinkmeaner's name comes up. Robert tries to reason with the Hateocracy to leave them alone and forget revenge, and they reveal they couldn't care less about the deceased Colonel- they're just ruining lives for the hell of it. Luckily, an army of cops shows up in the nick of time to cart off the unruly trio. Taking a jab of his own at his ungrateful cohorts, Stinkmeaner's ghost, who has been narrating on and off throughout the episode, hangs a cynical lampshade on his fate over his old pals: He thinks death and hell are better than being in jail!
Don King in Celebrity Deathmatch normally comes back from the dead after getting killed in the audience, usually on accident or by a monster. When he actually fought in a match, Donald Trump killed him, only to never be seen again.
The series Darkwing Duck has two villains who die (on screen): Splatter Phoenix, who melts while she gives a rambling speech as she's erased from existence, and Mayor Synapse who turns into a powerful almost indestructible being until the main heroes get him to blast and explode "graphically".
In Family Guy a few recurring characters such as Mr. Weed, Paddy Tanniger, Vern and Johnny, and Francis Griffin have been killed off. Despite Negative Continuity, any returns are made by their ghosts.
Joan Quagmire died on a technicality, having tried to grab Death's hand. As she was married to the "deceased" Glenn Quagmire and having taken his last name, he agreed to take her instead. Being a bit off didn't hurt, either....
Amusingly subverted with James Woods. He's seemingly killed off for good in that same episode only to turn up later on (complete with deliberately hilarious and absurd Voodoo Shark explanation for his return to boot).
Mental Mike in the Season 12 episode A Fistful of Meg.
In the Futurama episode "Benderama", a Patton Oswald-voiced giant nerd gets killed off by Bender and his many clones. Before he dies, he finally gets to know what it's like to be beautiful.
Webstor has the distinction of being the only member of the regular cast to truly die in the Masters of the Universe. In the "Web of Evil" episode of He-Man and the Masters of the Universe (2002), he was assaulted off-screen by the Snake-Men as the heroes were closing in, then brutally beaten by them and collapsing dead on the ground when the good guys finally reached his lair. However, the scene depicted his death in an ambiguous manner, so fans were left to debate themselves whether or not he had survived until after the show's cancellation, when Ian Richter confirmed that he did not. (It is entirely possible that the writers had planned for the possibility to reverse this decision and include him in a later story, seeing as the series was cancelled three episodes later.)
Justice League Unlimited killed off most of the Legion of Doom via an exploding spaceship in "Alive", the second-to-last episode of the series. While a small number were explicitly shown to survive, a number of minor villains like the Shade, Silver Banshee, Rampage, Neutron, and many others were presumed dead. Lex Luthor and Darkseid are apparently killed in the finale, "Destroyer", but Superman doubts that Lex is truly gone. Dwayne McDuffie has stated that Luthor and Darkseid now form part of the Source Wall, meaning they're technically alive, but unlikely to ever trouble the League again.
Warhok and Warmonga from Kim Possible were confirmed by Word of God to be dead for real after Ron used his Mystical Monkey Powers to throw them into their own ship after they threatened to kill Kim, after which the ship exploded. They're about the only villains in the series to be killed for real... Except perhaps for Monkey Fist.
In King of the Hill, quite a few recurring or main characters have been killed off over the years including Luann's first boyfriend Buckley, Buck Strickland's mistress Debbie, Pops Poppaseto, Cotton's war buddy Topsy, and most recently Hank Hill's father Cotton Hill.
Although there is possibility that Chrysalis fell to her doom, but nothing has officially confirmed this (and the comics, which some take as canon, depict her survival via Joker Immunity). Some also take Nightmare Moon to be a manifestation of Luna's bitterness possessing her, and seems to shatter to pieces after Luna is restored, but nothing in the episodes themselves suggests she's anything more than Luna corrupted. Therefore, Sombra is the first main villain to officially be Killed Off for Real.
G1 also had Lavan, who died in similar way to Sombra (although a little less graphically), after his magic beam ricocheted from the princess ponies' magic wands back at him.
In The Powerpuff Girls, this possibility arises with Dick Hardly, a one-episode villain of "Knock It Off", who, within the episode, cemented himself as probably one of the most evil villains on the show, may actually have been the only human villain to die, considering that, after his episode, him and his various PPG knockoffs (which were the ones that did him in) were never seen again.
Bunny in "Twisted Sister." The girls attempt to create a fourth Powerpuff Girl using substitutes for sugar and spice, and their subjective forms of everything nice, as well as an "accidental" dose of Chemical X. The product is an apparently mentally disabled sibling with a black and white sense of right and wrong, which causes her to not quite get the hang of crime fighting. The girls, once they find out what trouble she has been up to, tell her she did bad and cause her to flee in tears. The criminals that Bunny released as a result of viewing the police as bad in her black and white view of the world, attack and overwhelm the girls, and Bunny hears their cries and comes back to save them. The ensuing battle causes her already unstable form to reach its maximum capacity, and she explodes after proudly proclaiming that she finally did good. The narrator confirms she's not coming back.
Narrator: -tearfully- And for the first, and final time, the day is saved thanks to Powerpuff Bunny.
Parodied in an episode of The Simpsons. Moe gets a facelift and becomes handsome, and is awarded a role on a Soap Opera. After reading a new script that says he dies, he retaliates by revealing all of the major plot twists in the show for the next year on air (the show was filmed live, apparently). The director than angrily informs him that his character wasn't being killed off and that that page of the script was a dream sequence.
And not parodied with the very "real" (at least in animation terms) deaths of Bleeding Gums Murphy and Maude Flanders. Dr. Marvin Monroe was also apparently dead enough to have a hospital named in his memory (but he got better...) and Hans Moleman has technically died multiple times (due to explosion, motor accident, fire, a pack of hungry wolves, etc.) but has yet to have been killed off for real.
Dr. Nick Riviera was killed off in The Movie, but he got better and resurfaced later on the TV show.
When the hated Poochie is killed off, Krusty signs a legal document swearing that he will never, ever come back. And then he came back in a later Itchy and Scratchy episode as a cameo guest to one of Scratchy's funerals.
This trope finally caught up with Mona Simpson, Homer's mother. What makes this death particularly impacting is her successive Not Quite Dead track record in previous episodes.
Mob boss Fat Tony dies of a heart attack, but in a Subverted Trope, he was replaced by his identical cousin who also got fat.
Frank "Grimey" Grimes is dead and his son confirmed it.
Wrong. In the follow-up, Father Christmas, the Snowman (and Boy) are discovered alive and partying at the North Pole. The dialogue makes it clear that this is a year after the events of The Snowman. Raising the possibility that a dead Snowman can simply be resurrected. Fridge Horror?
Subverted with Ahsoka in the Season 3 episode, Altar of Mortis, where she is killed by the Son of Mortis (who was controlling her) after giving him the Dagger of Mortis. Ahsoka however doesn't remain dead unlike most of the other characters that are killed off, as she was brought back to life when Anakin transfers the dying Daughter of Mortis's life into Ahsoka.
At the end of the Superman: The Animated Series two-parter "Apokolips Now", after Darkseid's invasion of Earth is thwarted and just before he leaves he murders Superman's friend Metropolis Police Inspector Dan Turpin with his Omega Beams in front of Superman and a crowd of onlookers. Some fans hoped Turpin was just teleported to Apokolips like Kalibak had been after he got apparently zapped, but it seems he's gone for good.
Beast Wars continually killed its cast, despite the fact that Beast Wars characters are a bit hard to kill, as robots they have survived deadly attacks and being blown to bits (in which case they can easily be repaired). Some simply died, in one case to make room for new characters. A surprising number died, were brought back, and died again. Two of them died, were brought back as a fused form of the two, and then died. And two of them died with the possibility that they might return, but didn't.
Major characters to permanently die are (in order of death) Terrorsaur, Scorponok, Dinobot, Tarantulas, Rampage, Depthcharge, Tigatron, Airazor (both as Tigerhawk), Inferno, Quickstrike, and Dinobot II. Major characters to survive to the end of the final episode are Optimus Primal, Rattrap, Cheetor, Rhinox, Blackarachnia, Silverbolt, Megatron, and Waspinator.
If you count the comics, the list of permanently dead characters is reduced to both Dinobots, Rampage, Tigerhawk, Inferno and Quickstrike.
And the in the sequel series, Beast Machines, Optimus, Megatron, and Rhinox are killed as well.
In Transformers Animated Blurr is killed, pretty graphically, for a machine. Namely he was crushed into a cube and dumped down a trash chute.
The finale also killed off Prowl and Starscream. Prowl sacrificed his Spark to save the city, and Starscream's Allspark fragment, the only thing keeping him alive after he was killed by Megatron in "Megatron Rising", was pulled out of his head. To hammer the point home, their bodies turned completely grey, the telltale sign in this series that a Transformer is really dead barring interference from the Allspark itself.
Jazz was killed by Megatron in the final battle of the first live-action movie. The Fallen was killed by Optimus at the end of the sequel and every Decepticon (who isn't Megatron) that has been killed has stayed dead so far.
In Winx Club they have killed off the main villains of season 2, 3 and 4 at the end of those seasons. In addition to this, we lost Nabu in season 4.
W.I.T.C.H. killed off quite a few of the lesser villains, such as Tridart and Ember being disposed of by Nerissa when they were no longer needed. In the finale, Tracker is also subsequently impaled and killed.
Kent Nelson a.k.a. Doctor Fate. His spirit did reside in the Helmet of Fate for a while, but Fate eventually got tired of his nagging every time someone used the helmet and sent him off to the afterlife.
In the last episode, Kid Flash is disintegrated when he absorbs too much energy helping The Flash and Impulse get rid of an energy field that threatened to envelop the Earth. To honor his memory Impulse takes up his mantle. To hammer the point home, he gets a hologram in the garden like Jason Todd, Tula, and Ted Kord.