47. Donkey Kong (Donkey Kong) For: Being an early gaming villain and an nefarius one at that
46. The Butcher (Diablo) For: Sadism and overall unsavoriness; "Ah, fresh meat!"
45. The Collosi (Shadow of the Colossus) For: Being such a visually stunning and memorable villain
44. Baal (Diablo II and III) For: Murder, evil and being an unsavory character.
43. Kane (Command & Conquer)For: Murdering his brother
42. Dahlia Gillespie (Silent Hill)For: Being an antagonist in games where the town itself is your antagonist.
41. Lady vox (EverQuest)For: Pwning so many n00bish RPG players.
40. GLaDOS (Portal)For: Apathy and lying about the dispersal of cake.
39. Dr. Wily (Megaman)For: Killing Dr. Light and making a bunch of evil robots.
38. The Thief (Zork)For: Plagiarism
37. The Creator (FF Legends)For: Being an epic boss (he's GOD for pete's sake).
36. Ragnaros (WoW)For: Pwning MMORPG players, cuz' we all know they need a little pwning.
35. Officer Tenpenny (GTA: San Andreas)For: Being a dirty cop.
34. Lavos (Chrono Trigger) For: Being an evil boss and not dying.
33. Sephiroth (FFVII)For: Being angry at the world and attempting armaggedon.
32. M. Bison (Street Fighter)For: Being a villain in a fighting game.
31. Icon of Sin (Diablo II)For: Being the definition of diabolical.
30. Master Hand (Kirby Series, Super Smash Bros. Series)For: giving so many puffball players PTSD and the Smash power.
29. Jon Irenicus (Baldur's Gate II: Shadows of Amn)For: Sadism and being a devious villainish guy.
28. Sargeras (WoW)For: Corrupting a race of peaceful beings.
27. Lich King (WoW)For: Raising an army of the undead and trying to exterminate the humans.
26. Giygas (Mother and Earthbound)For: Being the physical embodiment of evil.
25. The Flood (Halo: Combat Evolved)For: relentles attack that leaves no time to even enjoy a light snack.
24. Nemesis (RE3: Nemesis)For: Causing so many cases of PTSD
23. Dark Falz (Phantasy Star series)For: See original list.
22. Darth Malak (SWKoToR)For: Trying to usurp the universe.
21. Prophet of Truth (Halo)For: Blackmail and attempted mass extinction.
20. Goro (Mortal Kombat)For: See original list
19. Arthas (WoW)For: Becoming a pawn of the Lich King and helping to topple the human capitol.
18. Ares (God of War)For: See original list.
17. Ridley (More or less any Metroid game)For: Killing Samus' parents and everyone she held dear and leaving her to live in the desolation.
16. The Cyberdemon (Diablo II)For: Being so freaking evil.
15. Dr. Robotnik (Sonic Series)For: Attempting to kill cute furry forest creatures on more than one occasion.
14. Diablo (Diablo)For: corrupting the original game's protagonist before the second game.
13. Sinistar (Sinistar)For: Making us scramble to stop the workers from assembling said weapon.
12. SHODAN (System Shock)For: Relentlessly taunting (Monty Python and the Holy Grail, anyone?) you through a massive computer system.
11. Dracula (Castlevania Series)For: Being relatively diabolical in the actual sense of the word.
10. Darth Nihilus (SWKoToR II)For: Trying to commit genocide and doing it while dressed as a freaking CLOWN (Claurophobia, anyone?).
9. Exdeath (A FF Game)For: Becoming a main antagonist in two FF games after being animated from a TREE.
8. Bowser (Mario Series)For: Putting Mario and Luigi through so much crap.
7. Kerrigan and the Zerg Overmind (Starcraft Series)For: Giving SCII veterans chills whenever they hear "Omega class energy signature detected. Queen of Blades inbound."
6. Mother Brain (Metroid through Metroid: Zero Mission)For: Killing an innocent baby Metroid and giving Samus, a badassery icon everywhere, PTSD as a result.
5. Kil'Jaedan (WoW)For: Forcing a peaceful person to become an icon of death and and destruction by threatening to make his soul spend the rest of eternity in torment.
4. Dark Samus (Metroid Prime Trilogy)For: Making the entire Space Pirate race declare war on humanity and and giving MPIII vets nightmares about having Phazon overloads and turning into it.
3. Ganondorf (Legend of Zelda Series)For: attempting (and succeeding occasionaly) to take over Hyrule about a million times.
2. Albert Wesker (Resident Evil Series)For: Backstabbing the STARS team in the original RE and faking his death at least four times since.
1. Kefka (Final Fantasy VI)For: being the only video game villain to actually succeed and becoming a GOD.

My descriptions of WHY they made the list are rather lacking, so please just focus on the placings of the characters rather than my why.
PS (n00bs only):
WoW is World of Warcraft
SC is Starcraft
MP is Metroid Prime
FF is Final Fantasy
RE is Resident Evil
PTSD is Post-Traumatic Stress Disorder
STARS is Special Tactics and Rescue Services
SWKotOR is Star Wars Knights of the Old Republic
GLaDOS is Genetic Lifeform and Disc Operating System
MMORPG is Massive Multiplayer Online Role Playing Game
GTA is Grand Theft Auto
And my signature pwns your signature any day of the week.