The home side took the lead in the 15th minute through Franculino Djú.
Mama Baldé doubled the advantage in the 66th minute.
Álvaro Djaló sealed the victory with a late goal in the fifth minute of stoppage time.
Nigeria entered the fixture as strong favourites after their previous win over Madagascar.
However, the Super Eagles struggled to create clear chances and failed to find the net.
Nigeria controlled more possession but were repeatedly caught out on the counter.
The result leaves Guinea-Bissau top of Group L with six points from two matches.
Nigeria sit second with three points from two games.
The Super Eagles have recorded one win and one defeat.
The result marks a difficult away night for Nigeria in the 2027 AFCON qualifying campaign.
It also raises questions about their consistency on the road against lower-ranked opponents.
