1.Glad0s (Portal)- I get the feeling everyone who played the portal series and made a list is going to be banging their head against the wall for not adding her now.

2. Xykon (Order of the Stick webcomic)- Few other villians reach the level of evil that Xykon is at, none manage to be so fucking comedic while doing so.

3. The Thing(John Carpenter's The Thing - If it existed, it would have taken over the universe by now.

4. Gentleman Johnny Marcone (Dresden Files)- He's pretty much always a side-protagonist, but he is definitely a villain. He's a normal human in a supernatural world who is so badass he has been recognized by major supernatural entities as the baron of Chicago.

5. Jan Valentine (Hellsing Ultimate, The TFS abridged Version) - Skull-fuckings for everyone!
 
Honorable Mentions:
Master (Fallout 1)
Pyramid Head (Silent Hill)
The Lich (Adventure Time)
SHODAN (System Shock series)
Clockwerk(Sly Cooper series)
Wheatley (Portal 2)- The Only reason he's not on the list is because I didn't want to fill it with multiple characters from the same series.
And most other villians people have listed