Another vote for the Fire Ants in Fallout 3.
And a rather nasty surprise in Oblivion. Oh, a cute little glowing thing. Which is immune to attacks from my steel broadsword. And I'm as thoroughly unmagical a character as you can possibly be in that game. I found out about 10 minutes later that one of my friends had also "tweaked the difficulty slider a little" to see what would happen. By "tweaked a little" he actually meant "set to maximum".
Also, I've caused a few of these moments for others in Homeworld.
Enemy sends a small party of bombers and multigun corvettes basically as a scouting raid. They charge in and get torn apart by missile destroyers before getting too close. Apparently he saw enough of my fleet to make a comment.
"You're not even trying, are you?"
As his attack fleet emerges from hyperspace, explosions rock them. His Heavy Cruiser fares best, the minefield only crippling it, instead of destroying it outright. My Ion Frigates quickly move in for the kill, though, careful not to come in range of the minefield which is still drifting towards his Cruiser.
He saw the frontal defensive line of my fleet - in Parade formation, no less (the least combat-effective formation you can possibly use, but makes it easy to pick out and identify ships). He thought I only had 2 Missile Destroyers and a swarm mostly made of Ion Cannon Frigates, with a handful of Assault Frigates for support. The minefield was set behind my fleet, and the Corvettes which planted it were docked in the Mothership, as a preventative against exactly the kind of attack he was attempting.
Earlier, I'd moved my own Heavy Cruiser a long way off in a random direction, with a Destroyer and a Missile Destroyer as escorts. These three ships came out of hyperspace a short distance from his Mothership, and launched an assault as his Heavy Cruiser exploded. Coming up from below, We caught him totally unprepared, a swarm of missiles eliminating his smaller craft as we moved in, his Mothership the only target once these were gone. He only had a handful of Assault frigates left to defend himself.