"are you currently or have you ever been a character of a computer game?"

"If you've come for the fork, you'll be sorely disappointed."

"The cake is a lie." (Sorry if it has been used, didn't feel like digging around.

"Never spend more than 20 bucks on a computer game."

"I'm not all smiles and sunshine!"

: D

"Dravis you son of a---IF you wish to decline further service you will be considered in default of your contract, and your fee may be suspended, pending litigation. Good luck material defender, Dravis out." --Descent 2

"Burn you monstrosity...burn..." --Homeworld: Cataclysm

"I start to breathe again." --Descent 1

Also the entire ending scene in Serious Sam is awesome! ;)

from diablo 2
Diablo: [after opening the last seal] Not even death can save you from me

from mgs 3
The Boss: I raised you, and loved you, I've given you weapons, taught you techniques, endowed you with knowledge. There's nothing more for me to give you. All that's left for you to take is my life.

from monkey island
"“That’s the second biggest monkey head I’ve ever seen!”"
"Guybrush: I'm on a whole new adventure.
Bart: Growing a mustache?
Guybrush: No. Bigger than that.
Bart: A beard?!?"
"Guybrush Threepwood: [singing] Oh... there's... a... monkey in my pocket / And he's stealing all my change / His stare is blank and glassy / I suspect that he's deranged! "

from Indiana Jones and the fate of Atlantis
"Indiana Jones: [while exploring the Crete labyrinth] Some date, huh?
Sophia Hapgood: We're not dating Jones; this is not a date, if it was a date, I would've stood you up! "
"I'm selling these fine leather jackets"

Full Throttle:
When I'm on the road, I'm indestructible. No one can stop me... but they try.
--
You know what might look better on your nose? The bar!

Star Wars Republic Commando:

Was it red-red-green, or red-green-red?

Magician Lord on the Neo Geo is full of comedy gold with your nemesis, Gal Agies:

http://www.youtube.com/watch?v=SOKRBCQTZDk

My favorite: "You are very dangerous, be dead down here."

If you're going to quote Portal, at least you should be quoting GlaDOS.

"I think we can put our differences behind us. For science. You monster."
"I'd just like to point out that you were given every opportunity to succeed. There was even going to be a party for you. A big party that all your friends were invited to. I invited your best friend the Companion Cube. Of course, he couldn't come because you murdered him. All your other friends couldn't come either, because you don't have any other friends. Because of how unlikeable you are. It says so here in your personnel file: Unlikeable. Liked by no one. A bitter, unlikeable loner whose passing shall not be mourned. 'Shall not be mourned.' That's exactly what it says. Very formal, very official. It also says you were adopted. So that's funny, too."
"The Enrichment Center is committed to the well being of all participants. Cake and grief counseling will be available at the conclusion of the test. Thank you for helping us help you help us all."
"And then there will be cake."

"Mike and Sean, f*ck yeah! ...with three exclamation points"

"Attention interloper, heed this recorded message! This drone-vessel speaks with the voice and authority of the Ur-Quan. You are trespassing within Ur-Quan space. This world, Earth, may not be approached for any reason. Nor will hostilities against our orbital platform be tolerated. In addition, your ship does not respond to standard Hierarchy identification transmissions and is therefore deemed to be independent. This is not permissible, only subservience shall be tolerated. This drone now leaves to inform the Ur-Quan of your transgressions. You are commanded to remain here and await the arrival of the Ur-Quan. Disobedience will be punished." - Ur-Quan Drone, Star Control II: The Ur-Quan Masters.

Epicus doomicus gamixus, i guess.

"Perhaps [it's] not common knowledge, but everything is plain for those that know where to look." - Elminster in Baldur's Gate 1.

NeverEnd :

" -As a reward, I'm giving you a tusk."
" -Huh, you can stick that tusk where the sun doesn't shine. Pahh! I want a proper reward, as promised."

"Sometimes I terrify myself with my creative genius."

"I'd successfully got the marker into a position near the top of the needle simply by using the kind of lateral thinking that can get you institutionalised"

Both are George in Broken Sword 2. I could have picked hundreds from that game.