Vilgax, after getting a valuable crystal, destroys the entire planet that it came from, Petropia, for no reason. The Petrosapiens (AKA the same species as Diamondhead and Tetrax) weren't doing any harm to him, and he fired on their planet for no reason other than to test just how powerful the crystal is, and shows no regrets. And worse yet he had hired a Tetrax, a Petrosapien bounty hunter, to aquire said crystal without telling him he would be destroying his own planet.
Zombozo gets his by kidnapping a 10 year old girl and draining her of her energy until she's nothing but a shriveled husk of her former self, speaking of it so lightly that it's implied it's not the first time.
Zs'Skayr AKA the evil version of Ghostfreak that was spawned when his mind escaped the Omnitrix in "Ghostfreaked Out" first crossed the line when in that episode, he threatened to kill a possessed Gwen if Ben did not surrender, fully aware that both are only 10 years old.
After being resurrected, Ghostfreak crosses the line further with his plan to plunge Earth into eternal darkness, and the result of the device being used to do so is shown to be horrifying mutations of every living thing on Earth. That was just a side effect of his plan that he didn't even care about.
Kevin 11,000, the first future version of Kevin 11 as an adult (from way back when it seemed unlikely that Kevin 11 could be redeemed, back before Ben 10: Alien Force came along to prove us wrong), crosses the line when he decides the best way to take revenge on Ben is to kill Ben's 10-year old son. And when Kevin's own son tries to stop him, he literally throws him aside and shows that he doesn't care about him at all, and then seems perfectly fine trying to kill him too when he fights back.
Forever King Driscoll's Establishing Character Moment from when he debuted at the end of "Perfect Day" literally has him crossing the line and proving himself more evil than Enoch, when he deliberately leaves Enoch to be trapped forever in his own Lotus-Eater Machine just to spite him for not getting the Omnitrix.
In Ultimate Alien, Aggregor did this onscreen by murdering five innocent aliens in cold blood. However, he could be considered to have already crossed it when he came up with a plan ending with him murdering a newborn.
Defying Villain Decay, Zombozo crosses this by forcing Ben's mother to walk a tight rope and then lighting it on fire underneath her, intent on either burning her alive or sending her falling to her death. This pushes Gwen so far that she assumes her One-Winged Angel form and scares the living crap out of him.
Colonel Rozum imprisoning 775 aliens in a horrible underground prison for over five decades, many of whom likely did nothing wrong. Prisoner 775 for example was exiled to Earth by an alien tyrant who he was trying to save his planet from. He told Rozum this, multiple times and they didn't listen. The only good thing about this guy is his love for his family.
Prisoner 775 himself has one. Once he escapes, he intends to exact his revenge against Rozum. A viewer might not condone it, but could certainly understand his reasoning for revenge against just Rozum. Problem is, 775's targets expand to Rozum's whole family. They had absolutely nothing to do with his captivity, and he knows it. He just wants to kill them to make Rozum suffer. This may have been an intentional crossing of this on his part, as in the end he attempts Suicide by Cop, so he may have just wanted to do something that would force Ben to kill him and end his suffering along with getting his vengeance.
Charmcaster sacrificing thousands of souls, even killing Ben, Gwen, and Kevin, in order to make a Deal with the Devil that will resurrect her dead father. Though to be fair, she does have a My God, What Have I Done? and go into a Villainous BSoD when he calls her out on it, and since this is a series where alien Nazis like the Highbreed can be forgiven...
Word of God comments made online actually seem to indicate that this act is, in fact, an INVERSION of the Moral Event Horizon. Instead of being the point where Charmcaster crosses the line into irredeemability, her realization of how wrong it was due to her father's words has pushed her completely AWAY from evil, and she is thus now a True Neutral character rather than still a villain.
Captain Nemesis crosses the line in "To Catch a Falling Star" by becoming the first successful hands-on murderer in the series. He kills several people, including a random motorist just to steal his car. Even his Pet the Dog moment at the very end doesn't bring him back across the line.
In Omniverse, Malware crosses the Event Horizon when he destroys his own homeworld, Galvan B, in the finale.
Years after he crossed it in the original series, Vilgax undergoes Character Rerailment in Omniverse in And Then There Were None, where he unleashes a bomb that erases all timelines save for the one he's in now, where its version of Ben Tennyson doesn't have an Omnitrix. Essentially, he's wiped out entire worlds, wiped out a number of lives too great to count, including his own allies, even erasing the main timeline's Ben Tennyson from existence, all to exist in a world where he can easily overcome a Ben Tennyson who can't fight back and rule unopposed.
True Neutral character rather than still a villain.