Orlando Pirates striker Justin Shonga put the Chipolopolo ahead with an exquisite free kick in the 11th minute after Enock Mwepu had been brought down on the edge of the penalty box.
A revived Guinea Bissau put back their campaign on track with a Stoppilla Sunzu own goal and Tony Silva header that handed the host nation a vital three points.
Guinea Bissau surged to the top of the table with seven points.
Zambia will now have to revert to ‘calculator style’ antics to negotiate qualification with only group leaders automatically qualifying to the 2019 Africa Cup of Nations.
The Chipolopolo boys won 2-1 on Wednesday and had set a minimum of a draw as a target to keep their hopes alive.
Zambia’s next match is an away fixture to Mozambique in November.