A file photo showed Nigeria's Victor Osimhen who missed the game against Guinea due to injury. AFP
Atromitos Athens midfielder Aguibou Camara opened the scoring for Guinea in the 14th minute of the game.
Nigeria's Simon Moses had an opportunity to equalize for the Super Eagles, but the Nantes FC winger missed a penalty just five minutes later.
Guinea extended their lead in the second half, with Facinet Conte finding the back of the net four minutes after the hour-mark.
Nigeria's African Player of the Year, Victor Osimhen, watched the game from the bench as he is yet to recover from a minor injury.
Nigeria, three-time African champions, have been placed in Group A alongside hosts Cote d'Ivoire, Guinea Bissau, and Equatorial Guinea.
Meanwhile, Guinea will compete in Group C against Cameroon, defending champions Senegal, and Gambia.
The Africa Cup of Nations will take place in Cote d'Ivoire from 13 January to 11 February.
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