Video games have plenty of lady artificial intelligences (hey, Cortana) including megalomaniacs (hey, Shodan), but GlaDOS from Portal is memorable even in a crowded field. It's the sense of humor that does it, the streak of playground malice that compels her to make fun of you while subjecting you to dangerous experiments.
Iconic moment: Even as you pull GlaDOS apart she continues insulting you, explaining that none of your friends would come to your party, "Because of how unlikeable you are." Then she pretends to read your file. "Unlikeable. Liked by no one. A bitter, unlikable loner whose passing shall not be mourned."