Franculino Djú, Mama Baldé and Álvaro Djaló scored for the Djurtus, who followed up their opening 2–0 victory over Tanzania with another three points to move clear at the top of Group L.
Nigeria entered the match after recovering from a goal down to beat Madagascar 2–1 in Uyo, but Éric Chelle's side struggled to reproduce that performance in Bissau.
Djú gives Guinea-Bissau the lead
Guinea-Bissau made the stronger start and broke the deadlock in the 15th minute.
Franculino Djú created space before curling a fine effort into the far corner, leaving Nigeria goalkeeper Stanley Nwabali unable to keep out the opener.
Nigeria attempted to respond, with Ademola Lookman providing an attacking threat, but the Super Eagles struggled to create clear-cut opportunities before half-time.
Chelle made two changes at the interval, introducing Moses Usor and Moses Simon as Nigeria looked for a way back into the contest.
Baldé doubles the advantage
Nigeria showed more attacking intent after the restart, but Guinea-Bissau remained dangerous on the counter.
The hosts doubled their lead in the 66th minute through Mama Baldé.
The forward cut inside from the left before sending his effort into the net, leaving Nigeria with a two-goal deficit. Baldé had also scored in Guinea-Bissau's opening victory over Tanzania.
Chelle responded by bringing on Tolu Arokodare, while later substitutions saw George Ilenikhena and Raphael Onyedika introduced as Nigeria searched for a response.
However, the Super Eagles could not find a breakthrough.
Djaló completes the rout
Guinea-Bissau added a third goal in the fifth minute of stoppage time.
Álvaro Djaló finished from close range to complete a 3–0 victory and seal Nigeria's first defeat of the qualifying campaign.
The result also marked Guinea-Bissau's second victory over Nigeria in AFCON qualifying, following their famous 1–0 win in Abuja during the 2023 qualifying campaign.
Nigeria now face a battle in Group L
The defeat leaves Nigeria on three points from two matches, while Guinea-Bissau move to six points at the top of Group L.
The qualification situation is unusual because Tanzania are already guaranteed a place at the 2027 AFCON as one of the tournament's co-hosts. That means the remaining teams in Group L are competing for the additional qualification place.
Nigeria had opened the campaign with a 2–1 comeback victory over Madagascar, with debutants George Ilenikhena and Moses Usor scoring after Ehsan Kari had given Madagascar the lead.
But in Bissau, the Super Eagles were unable to produce another comeback as Guinea-Bissau punished Nigeria's defensive lapses and claimed all three points.
Guinea-Bissau 3–0 Nigeria
• Guinea-Bissau: Franculino Djú 15', Mama Baldé 66', Álvaro Djaló 90+5'
• Nigeria: No goals
Nigeria's next fixture: The Super Eagles are scheduled to face Russia in an international friendly on 6 October 2026 in Nizhny Novgorod.