There are just so many bosses from single player games that gave me trouble near the end of my experience with them that it's sort of a toss up. I'd say the Final boss of FFXII Revenant Wings on 100% completion. The hardmode fight comes literally out of no where, as there is no warning that 100% completion activates the harder fight, and the boss has a level 99 summoned monster walking around one shotting anything that it crosses paths with.
Now MMOs? Hands down the hardest was No Light Yogg Saron. I think a lot of raiders thought they had seen the worst raiding had to offer after making it through Fire Fighter on the Mecha Gnome area. When people hit no light, well... that just got ugly rather fast. Only a handful of guilds actually managed to complete the challenge mode when it was at it's highest difficulty, and even after the raid got nerfed it still gave lots more guilds a run for their money.
Now MMOs? Hands down the hardest was No Light Yogg Saron. I think a lot of raiders thought they had seen the worst raiding had to offer after making it through Fire Fighter on the Mecha Gnome area. When people hit no light, well... that just got ugly rather fast. Only a handful of guilds actually managed to complete the challenge mode when it was at it's highest difficulty, and even after the raid got nerfed it still gave lots more guilds a run for their money.