Whoever mentioned the ladder scare from F.E.A.R:
Oh. My. God.
I swear to god I shut down my computer and my screen as I was nearing the end of the ladder, I just startled.
The memorable intro from Bioshock comes to mind, when you're helpless inside that...the...submarine-round-thing-with-lights-and-incredibly-fragile-wires-apparently-thingy. I can't, for the life of me, recall the name of that thing. I got over it 2 minutes in, and the game stopped being scary from then on. Still pretty atmospheric though.
There was some weird audio glitch in Left 4 Dead. I was playing Co-Op on Expert on Blood Harvest (it was right around that part where you have to start the train so the bridge gets knocked down, I think it's the mission before the finale). Anyway, those who played that might or might not recall some sort of a shack or barn, painted red. We were all just sprinting in front of it, when I decide to go check in the barn to see if any Molotovs' had been left there by careless other survivors. Anyway, as I go in I just run in, my flashlight was turned off 'cause there had been a Witch earlier and I forgot to turn it back on, I pick up a molotov and turn right around. Boom! Right in front of me, a damn TANK! I had not heard him, nor had the music started playing when he started following us. I just turned around and he was there. Needless to say I think I woke up my neighbors screaming on my microphone for support.
EDIT: I thought of two more actually:
When I was 5 or 6, I remember being scared so bad by those Opera-singing and Cuddling zombies in Ocarina of Time. The truly funny part was in Majora's Mask near the 4th temple, I remember seeing the exact same zombies. I was like: You're kidding me right? So I never finished that temple...
Those damn regenerators in Resi 4. Ugh. Especially when you first see him and you have to enter this room right next to him. And then the freezer room. And then the spiked one that you see on a camera but can't figure out where he is. And then that garage-thing where they both pop out of nowhere, twice!