Well, I saw the title and thought “Keeel moose and squirrel!”
Upon further thought, however, I’m sad that I’ve been beaten more than once to Livia. It’s beautiful how she makes banal little Messalina from the sequel seem like such an amateur.
Zillah from Margaret Atwood’s The Robber Bride. Great book, great villian.
Joan Crawford in Mildred Pearce.
Cruella DeVille even has her own theme song.
I offer Norma Desmond from Sunset Blvd.
I was going to start this thread one of these days, if only to mention Grandma from Preacher. If graphic novels are allowed, she is my nomination. What a bitch.
The movie is pretty faithful to the book, but if you enjoy the movie portrayal of Annie, I’d really recommend you read the book. She is pathetic, repulsive, simultaneously caring and sadistic. Wonderful villain.
From GI Joe, The Baroness, a bespeckled, leather-catsuit-clad, Eastern European accented, terrorist leader.
Comes across as a revolutionary polemical-spouting cross between a schoolmistress & a Dom.
Expert sniper.
Interrogator, & enjoys it.
Fighter pilot, & loves to strafe unarmed civilians.
Past mistress of betrayal, treachery, & coup-de-tas.
Mistress of Disguise.
Pats you on the back until a bullet hole appears between her fingers.
And yet she was the subject of so many of my prepubescent fantasies! 
The Alien
“Olivia” from Peter Straub’s novel Julia. Revolting, and all the more horrific because she’s a child.
Ummm, she wasn’t the villian, she was the heroine. Her daughter Veda was the villian . . .
. . . and since I managed to spell “villain” wrong twice, I won’t bring up your spelling “Pierce” wrong . . .
the Queen / old crone in “Snow White.” (Disney version, and the Sigourney Weaver version).
Alex (Glenn Close) from “Fatal Attraction.”
Tony’s mother Livia (Natalie Marchand) from “the Sopranos.”
Norman’s (Anthony Perkins) mother from “Psycho.” (Yes, by the time of the events of the movie, she’s already dead. But considering Norman’s condition, she must have been a truly evil bitch.)
From the bible: Jezebel (high priestess of Babylon’s Baal cult), Delilah (the harlot who betrayed Sampson).
From greek mythology: Hera, Medea, Pandora (somewhat sympathetic, a tool of the Gods, but still…), Calypso, Eris (goddess of discord, sets in motions that lead to the Trojan war), Clytemnestra…
From “Star Trek: TNG” - the Borg Queen
I’ll second Annie, and tell you the creepiest thing about seeing the movie version–Kathy Bates, in that film, looks exactly like my mother. I saw it in the theater, and have never watched it since.
Dolores Umbridge from Harry Potter and the Order of the Phoenix. One of the best love-to-hate-her villains I’ve seen in years.
Diana, the alien leader from V. She always scared the crap out of me when I was a kid.
If we’re going with '80s cartoons, then Evil-Lynn from the He-Man/She-Ra franchise ought to count. Heck, she’s even got “Evil” in her name!
(Though I confess I don’t remember if she was He’s villianess, or She’s…)
Ooh yeah. She’s deliciously evil.
Mona Demarkov from Romeo Is Bleeding.
He’s He-Man’s villain. I recall quite clearly one of those “we all have to get along” episodes where she got stuck in the desert with Teela and they had to cooperate to survive.
Sigh. She is in He-Man. Duh.
I see some TV characters in here, so I’m going to nominate Sherry Palmer from 24. She had me screaming at the TV, “Shoot the bitch! SHOOT HER!!!”