Too many favourites of mine. I'll try to avoid quotes that have already been said.
"So, wake up, Mr. Freeman. Wake up and smell the ashes."
G Man - Half Life 2
"Would you kindly?"
Atlas - Bioshock
"My strength is not in steel and fire, that is what the parasites will never understand. A season for all things! A time to live and a time to die, a time to build... and a time to destroy!"
Andrew Ryan - Bioshock
"At the moment of synapse, as I teleport, this chamber will be bathed in deadly particles that have yet to be named by human science. Perhaps when I have the leisure to do the work myself, I'll name one after you. That way you won't be completely forgotten. When the singularity collapses, I will be far away from here. In another universe, as a matter of fact. You, on the other hand, will be destroyed in every way it is possible to be destroyed-and even in some which are essentially impossible."
Dr Breen - Half Life 2
"This isn't a war ordinary humans can win! This is the future - death's an inconvenience now, nothing more. We are all dead man walking."
Jacob Hargreave - Crysis 2
"It is one thing to forgive a slap across my cheek, but an insult to the Lord requires... no, it demands correction."
Joshua Graham - Fallout New Vegas: Honest Hearts DLC
"Our directives must be reassessed."
CABAL - Command and Conquer Tiberian Sun: Firestorm
"It would be a sad error in judgement, General Solomon, to mistake me for a corpse."
AND
"If I am cut, do I not bleed?"
Kane - Command and Conquer Tiberian Sun
"Well done. Here are the test results: You are a horrible person. I'm serious, that's what it says: A horrible person. We weren't even testing for that."
GLADOS - Portal 2
"Now you might be asking yourself, "Cave, just how difficult are these tests? What was in that phonebook of a contract I signed? Am I in danger?" Let me answer those questions with a question. Who wants to make sixty dollars? Cash. You can also feel free to relax for up to twenty minutes in the waiting room, which is a damn sight more comfortable than the park benches most of you were sleeping on when we found you. So! Welcome to Aperture. You're here because we want the best, and you're it... Nope. Couldn't keep a straight face."
AND
"Science isn't about why, it's about why not. You ask: Why is so much of our science dangerous? I say: Why not marry safe science if you love it so much? In fact, why not invent a special safety door that won't hit you in the butt on the way out, because you are fired!"
Cave Johnson - Portal 2
"Get out of here stalker!"
Random soldier - Stalker: Shadow of Chernobyl
"Father, do you not see? I control all of this. If I want to live, I live! If I want to take, I take! If I want you to die, you die!"
Rodrigo Borgia - Assassin's Creed 2: Brotherhood
"We can afford all we need, we cannot afford all we want."
President John Henry Eden - Fallout 3
"WE...OWN...YOU!"
The Keeper - Prey