Nigeria lost 1-0 at home to Guinea-Bissau on Friday in one of the shocking defeats in Africa Cup of Nations qualifying history.
Mama Balde, who plays for top French club Troyes, scored on the 30 minutes to put Djurtus (Wild Dogs) ahead in Group A.
Dalcio Gomes should have added a second in the 36th minute when he had the better of both Uzoho and Iwobi, but his shot went into the side netting
Nigeria, whose line-up included prolific Napoli scorer Victor Osimhen and are 32nd in the FIFA rankings, were firm favourites to win at home and extend their advantage to five points.
The referee added five minutes of stoppage time but Guinea Bissau held on for a famous win to take over the top spot in Group A with seven points from three matches.
The teams meet again in Morocco on Monday with the Eagles seeking quick revenge.
