Monster X from Cave Story. I'm playing the game for my first time on the 3DS (courtesy of the recent eShop download), and... My God. I had to use my only health potion and I still only beat that thing by the skin of my teeth. They could have just named this thing That One Boss, and it would completely deserve it.
Oddly enough, TV Tropes namedrops a certain other boss (I don't want to spoil anything...) above Monster X. That boss was not even HALF the fight Monster X was; in fact, it was surprisingly easy for a Cave Story boss. All I had to do for that one was jump and shoot a lot; the hardest thing about fighting Monster X was the co-ordination and aiming required even as you're dodging a hail of... tiny homing robots. *shudder*
Plus, the best part about the Monster X fight? There's hardly any build-up or warning of any kind, and Monster X has absolutely no relevance to the plot. It's a Giant Space Flea From Nowhere.
Monster X: An especially punishing fight presented by an already very challenging game.