Northern Ireland have only one chance remaining of making it to the World Cup finals after this setback at a stunned Windsor Park.
They go to the Czech Republic, knowing a victory in their final qualifier will be needed next month if a place in the play-offs is to become possible.
Goals from Stanislav Sestak and substitute Filip Holosko either side of half-time saw Slovakia cement their place at the top of Group Three.
Northern Ireland slipped down to third in the table on goal difference behind Slovenia and it was a disappointing performance, given the way they had picked up a point against Poland at the weekend.
It was also the first home defeat suffered in seven competitive games by Northern Ireland and as such unexpected.
The visitors looked dangerous on the counter-attack and surged forward after 13 minutes but Jonny Evans got the better of Robert Vittek as he prepared to shoot.
Northern Ireland, though, were rocked back on their heels two minutes later when Slovakia made the breakthrough.
Manchester City winger Vladimir Weiss, son of the manager of the same name, skipped past Evans and laid the ball into the path of Sestak, who made no mistake from close range.
Slovakia made it 2-0 in the 66th minute following a swift counter-attack with substitute Holosko knocking the ball past Maik Taylor two minutes after stepping off the bench.
Substitute Niall McGinn almost pulled a goal back in the 71st minute, only for his effort to clip the angle of post and bar.