Yes Man in Fallout: New Vegas is probably the funniest character of all time in my opinion. Can't remember any specific quotes off the top of my head, but the way he mixed suck-ups, salesmen and optimists into one disgusting personality is so delicious.

NOFL is chock full of those things. That game had me instinctively wait behind corners before attacking just to hear everything the NPCs had to say to each other.

Two quotes I recall making me laugh recently.

"It's a trap" in UnEpic
"It's a-me, Mario!" in Assassin's Creed 2

Borderlands 2: Mal sums up humanity pretty well ;)
https://www.youtube.com/watch?v=EkhBNbPPGd0

Pretty much everything Crassius Curio said in Morrowind. After leaving that mission in Vivec, I felt dirty... used... I needed someone to talk to about being touched on my bathing suit area.

Minsc is the winner for me. Everything he says is hysterical. "GO FOR THE EYES, BOO! GO FOR THE EYES!" Or my all-time favorite: "Camaraderie, adventure, and steel on steel. The stuff of legend! Right, Boo?"

A recent one - Shadow Warrior 2013, fortune cookie:

"To Be Is To Do - Socrates
To Do Is To Be - Sartre
Do Be Do Be Do - Sinatra"

Assassin's Creed II while parkouring in front of people - "He must be very late, and she must be very pretty."

Icewind Dale random spiders occasionally say something like - "What, you don't think all we say is eeeee do you?"

XCOM using the Arc Thrower - "1.21 gigawatts, baby!"

Dawn of War, Chaos Warriors..

Runner up -Sanity if for the weak!
First Place - The Weak fight back!

Always amuses me.

Minsc has a lot of funny lines in Baldur's Gate, and so does Xzar:
"I ate his liver with a nice chianti and some fava beans."
"I am become Death, destroyer of worlds!"
"Stop touching me!" and "Mommy, I don't feel so good."

and Jaheira has a nice line to Xzar:
"You are amusing in a 'what the hell is wrong with you' kind of way."

Chrono Trigger trial scene: Crono is put on trial for "premeditated abduction of royalty", hilarity ensues. Every little thing you did wrong in the game so far is brought up as "proof" of just how bad of a person you are.
The evil chancellor who plays the prosecution role in this monkey trial is particularly entertaining.

Here's a screencap of my favorite line of his:

Especially in their no-feeling-whatsoever-just-pure-fact voice.

Hah, reminds me of the loudspeaker annoucements in Claptrap's Robot Revolution (Robolution?) DLC.

"Is not a Claptrap entitled to the oil of his own brow? No, says Hyperion, it belongs to its owner! No, says the treasure hunter, it belongs splattered on the floor! But we chose differently. We chose...Robolution."


Also in Warcraft 2: Beyond the Dark Portal, when you click too often on some of the units:

Elven hero (who was female): "You don't touch the other elves like that!"
Death Knight: "WHEN MY WORK HERE IS FINISHED, I'M COMING BACK FOR YOU"

Well, pretty much every word in NOLF.
I really like some of the enemy shouts...

"Hard rain is falling, everyone down!"
"Bullets are not my favorite!"
"I don't care if it is friendly fire, I don't want any of it!"

Yes! I just started playing Morrowind after completing both Oblivion and Skyrim. When I finally saw the play he wrote I died.