At the start of the original Portal, GLaDOS is the computerised voice that guides you through the strange, alien environment of the Aperture Science facility. It quickly becomes clear that GLaDOS is unpredictable and insane, as well potentially murderous. This clarity comes as you start to realise that when it comes to your existence in the facility, she is God.
The relationship you develop with GLaDOS is an interesting one, hating her yet depending on her, even as she seems to depend on you. This is amplified all the more in Portal 2, which shows firstly a revived GlaDOS, and then a vulnerable GLaDOS who needs your assistance.
This godlike presence and refreshing vulnerability, along with the excellent relationship-building and dialogue makes GLaDOS my favourite antagonist in any game.
It does help that the dialogue and voice acting are both excellent:
And of course, she can sing:
My runners up in this category would probably be Monkey Island‘s LeChuck and Handsome Jack from Borderlands 2.