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A point would have seen Cotonsport reach the final four with a game to spare, but they may now need a positive result against Real Bamako on matchday six.
If Bamako are unable to beat ASEC on Sunday, Cotonsport will progress, though the manner of Saturday's defeat - their first in 17 matches - will have troubled coach Didier Gomes da Rosa.
Kader Bidimbou was Leopards' hero as his hat-trick helped the Congolese side cement second place in Group A.
Cotonsport captain Nicaise Zimbori-Auzingoni saw his penalty just before the hour saved by visiting goalkeeper Lawrence Ngome, who also kept out Daouda Kamilou's follow-up.
Bidimbou then struck twice in eight minutes to complete a hat-trick and secure a dominant win for Leopards.
Billy Mwanza's penalty early in the second half was enough to lift the Zambians into second place in Group B after Etoile Sahel went down 1-0 at home to Ivorian side Sewe Sport.
