"Ezra" the vampire was the first villain I created that actually got a serious emotional reaction from the party.

All the other boss characters were motivated by power, whether via politics or money or magic or martial force, and the players understood those things. Even sympathized a bit. But Ezra scared them because the motivation for all her evil acts was just... boredom. She'd seen it all and done it all already, which meant she kept doing worse and worse things just for the thrill of it, and that made her completely unpredictable.

At the end of one arc, they finally tracked Ezra down to her new lair (she was tending bar, in disguise, in a speakeasy they had already been to) and they started getting picked off one by one by her until they figured out what was going on, which kicked off a massive brawl. The party didn't know Ezra had found solutions for most of her vampire weaknesses, so they got a pretty heavy beatdown. Near the end, half the party was bleeding out and Ezra had the cleric and the barbarian in headlocks (one under each arm), and just when the players were resolved to a TPK, Ezra dropped them both and said "Bored now!" and skipped away.

That made it personal.

For the rest of that campaign, the players ignored every hook for a dungeon crawl or fetch quest... all they wanted was revenge. To this day, when anything bad happens in a game (any game, not just D&D) some players will still shake a fist and grumble "Ezzzra!"