That’s really tough, Glados and Wheatley are both great, along with other one like metal face and maruki. I would have to say my favorite as of right now is Tohru Adachi. He’s basically inverse yoshikage Kira
Kreia, or Darth Traya from Star Wars: Knights of the Old Republic 2: The Sith Lords.
Kreia might not technically* be a villain, but by the end of the game, either way, you must confront and defeat her, so I'd say that counts well enough.
[spoiler]* Take that as you will, but I recommend a grain of salt at minimum. She's a very complicated character and I feel like I cannot do her justice without diving into an overly long explanation of the game in general just for [i]context[/i]. Suffice to say, if you EVER get the opportunity to play SW:KOTOR2:TSL, I wholeheartedly recommend it. Even without the Restored Content Mod, it's an absolute masterpiece of a game.[/spoiler]
Do I need to do another write up on why SHODAN is categorically the greatest game villain of all time or should I just accept you're all too dumb to know better?