1) Wheatley (Portal 2)
2) Doopliss (Paper Mario TTYD)
3) Pegasus (Yu-Gi-Oh, first season)
4) Cartman (South park)
5) Derek Simmons and Carla Ramades (Resident Evil 6)

Honorable Mentions:

Calypso (Twisted Metal)
Oogie Boogie (Nightmare before Christmas)
M. Bison (Street Fighter series)
Mitochondria Eve (Parasite Eve)
GLaDOS (Portal)
Mr. Dark (Ray Man)
Veigar (League of Legends)