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.
Yet they could have gone ahead inside 60 seconds. David Healy tested goalkeeper Jan Mucha after Martin Paterson played the ball back to him.
Slovakia responded immediately and gave a taste of what was to come with Stephen Craigan having to make a crucial challenge on Sestak.
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.
Northern Ireland hit back in the 27th minute when Grant McCann picked out Healy but he sent his effort wide of the post.
It was a frustrating period for the province as they struggled to break down the visitors.
However they raised the tempo in the second half and McCann delivered a cross into the path of Damien Johnson but he was off-balance and headed over the top in the 48th minute.
But at least his effort got the crowd going and Paterson homed in on goal two minutes later after getting away from Martin Skrtel.
However Mucha raced off his line to make the block and deny the striker the equaliser.
This was a good spell by Northern Ireland and Slovakia were suddenly looking a bit vulnerable at the back.
Johnson then found space down the right in the 63rd minute but his cross only found the arms of goalkeeper Mucha.
Paterson again came close two minutes later after being picked out by Healy on the left but Mucha reacted superbly to beat away his shot.
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.
It was an uphill task now for Northern Ireland as they entered a crucial phase of the game.
Substitute Niall McGinn almost pulled a goal back in the 71st minute, only for his effort to clip the angle of post and bar.
Northern Ireland kept pushing forward but were unable to break Slovakia's resistance and Marek Sapara almost added a third, but his shot hit the post after he had stepped off the bench.
The defeat, their first in six qualifiers, leaves the province's hopes of booking their passage to South Africa hanging in the balance.