The final level of Unreal Tournament 2004 (Malcom/Xan/Clanlord).
I've spent so many hours in this game, definitely my favourite game of all time, but Jesus Christ, this level. The game has a pretty good difficulty curve until you get to that level and then it goes through the roof. In the original version of the game, the onlt way I could beat it was to avoid whichever opponent it was, avoiding fighting them at all costs until the time ran out, because they would inevitably commit suicide on their own at least once. In the custom edition, there's no time limit and I simply cannot do it. I can play the whole game up to that level on the 5th difficulty (Skilled I think it's called?), but I can't do the final level on the easiest difficulty.
Actually now I think of it, I couldn't do the final level of UT2003 either, but I haven't spent nearly as much time in that, and it had the added issue of weapons disappearing when you pick them up, so you were screwed if you died once, whereas the AI never seemed to have an issue finding more weapons or killing you with the ones you spawn with.
Also, not really a boss, but the sequence in prototype 1 where you have to defend that machine that's pumping something into the ground (before you fight Elizabeth Green as a massive blob monster), on hard mode. I found a glitch to make myself invulnerable (using critical mass abilities still reduced your health, but you could still regain it and enemies couldn't damage you), and I still couldn't keep that machine alive.