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Culled from across the pop culture spectrum: movies, television, novels, video games, and (of course) comic books. This being a comic book publication, naturally, the comic-related entries tend toward the front. Have at it!
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100. Mr. Joshua (Lethal Weapon)
099. Beholder (Dungeons & Dragons)
098. The Others (LOST)
097. Francis Buxton (Pee-Wee's Big Adventure)
096. Sephiroth (Final Fantasy VII)
095. Stewie Griffin (Family Guy)
094. Brainiac (Superman: TAS)
093. Norman Bates (Psycho)
092. Dean Wormer (Animal House)
091. Auric Goldfinger (Goldfinger)
090. Gentlemen (Buffy the Vampire Slayer)
089. Bill Lumberg (Office Space)
088. Frank Booth (Blue Velvet)
087. Endo (Lethal Weapon)
086. Mr. Blonde (Resevoir Dogs)
085. Colonel Kurtz (Apocalypse Now)
084. Deathstroke (DC Comics)
083. The French Taunter (Monty Python and the Holy Grail)
082. Luther (The Warriors)
081. Angel Eyes (The Good, the Bad, and the Ugly)
080. Clubber Lang (Rocky III)
079. Cancer Man (X-Files)
078. R.J. Fletcher (UHF)
077. The Flood (Halo: Combat Evolved)
076. The Socs (The Outsiders)
075. Big Brother (1984)
074. Herr Starr (DC/Vertigo Comics)
073. Biff Tannen (Back to the Future)
072. Tony Montana (Scarface)
071. Walter Peck (Ghostbusters)
070. The Balrog (LotR: Fellowship of the Ring)
069. J.R. Ewing (Dallas)
068. Megatron (Transformers)
067. Venom (Marvel Comics)
066. Sideshow Bob (The Simpsons)
065. Kevin (Sin City)
064. Syndrome (The Incredibles)
063. Samara (The Ring)
062. Gollum (LotR: Return of the King)
061. Johnny Lawrence (Karate Kid)
060. Judge Doom (Who Framed Roger Rabbit)
059. Bob (Twin Peaks)
058. General Zod (Superman II)
057. Principal Rooney (Ferris Bueller's Day Off)
056. John Doe (Se7en)
055. The Overlook Hotel (The Shining)
054. Leatherface (Texas Chainsaw Massacre)
053. Cobra Commander (GI Joe)
052. Kid Miracleman (Eclipse Comics)
051. Catwoman (DC Comics)
050. Max Cady (Cape Fear)
049. Amanda Woodward (Melrose Place)
048. Iago (Othello)
047. Bill the Butcher (Gangs of New York)
046. Starscream (Transformers)
045. Mr. Burns (The Simpsons)
044. Godzilla (Godzilla: King of the Monsters)
043. Serpentor (GI Joe)
042. Keyser Soze (The Usual Suspects)
041. HAL 9000 (2001: A Space Odyssey)
040. Buu (Dragonball Z)
039. Michael Corleone (The Godfather: Part II)
038. Dark Phoenix (Marvel Comics)
037. Predator (Predator)
036. Alex DeLarge (A Clockwork Orange)
035. Roddy Piper (WWE)
034. Mayor Richard Wilkins (Buffy the Vampire Slayer)
033. Tony Soprano (The Sopranos)
032. Hans Gruber (Die Hard)
031. Grand Moff Tarkin (Star Wars)
030. Dracula (Monster Squad)
029. T-1000 (T2: Judgement Day)
028. Jabba the Hutt (Star Wars: Return of the Jedi)
027. Hillbillies (Deliverance)
026. Jason Voorhees (Friday the 13th)
025. Ozymandias (DC Comics)
024. Agent Smith (The Matrix)
023. Darkseid (DC Comics)
022. Thanos (Marvel Comics)
021. Red Skull (Marvel Comics)
020. Khan (Star Trek II: Wrath of Khan)
019. Green Goblin (Marvel Comics)
018. Galactus (Marvel Comics)
017. Magneto (Marvel Comics)
016. The Thing (John Carpenter's The Thing)
015. Pennywise (Stephen King's IT)
014. Freddy Kreuger (Nightmare on Elm Street)
013. Aliens (Aliens)
012. Sauron (Lord of the Rings Trilogy)
011. Darth Vader (Star Wars)
010. The Shark (Jaws)
009. Pinhead (Hellraiser)
008. Lex Luthor (DC Comics)
007. The Borg (Star Trek: The Next Generation)
006. Hannibal Lector (Silence of the Lambs)
005. Zombies (Dawn of the Dead 2004)
004. Doctor Doom (Marvel Comics)
003. Palpatine (Star Wars)
002. Pazuzu (The Exorcist)
001. The Joker (DC Comics)
Got to give it up to my man the Joker, who went from a one shot villian to probably.. well according to this list.. the greatest fictional villian of all time.