That One Boss:
Krauser in RE5 on Professional
before you realise that knifing him is more effective than a whole magnum clip.
The one-on-one fight with the sniper in Vendetta in World at War, on Veteran (not a 'boss', but it's still one dude making your life hell
)
Sarevok on Baldur's Gate (PC), this is on the middle difficulty, I wouldn't even try it on Insane.
Carry Armor (I think that's its name) in FF7. I'm amazed no one else has said this, Ruby and Emerald are one thing, but they're optional, this thing isn't.
The Butcher in Diablo, as many people have said, when you're new to the game and don't know the cheap way to kill him
Metal Gear Rex in Metal Gear Solid
The One Level:
The Snatch and Grab Special Op on MW2 on Veteran. Harder than the Echo ones O_o
The last level of Super Mario Land 2, in the castle