Zambia were made to pay for poor finishing as Yassine Chikhaoui's 89th-minute winner gave Tunisia a come-from-behind 2-1 victory.
Honour Janza's side were good value for their lead after Southampton striker Emmanuel Mayuka broke the deadlock on the hour in Thursday's Africa Cup of Nations Group B clash.
But Mayuka and his attacking foil Rainford Kalaba could not add the crucial second, and Zambia were punished as Tunisia struck twice in the final 20 minutes in Ebebiyin.
Ahmed Akaichi drew them level, before Chikhaoui completed an unlikely comeback with a late header.
Both managers made three changes to the starting line-ups from their opening games, with Tunisia's goalscorer in their 1-1 draw with Cape Verde, Mohamed Ali Moncer, dropping to the bench.
But Tunisia did not appear short of ideas in attack during the opening minutes.
Akaichi and Wahbi Khazri were worrying Zambia's defenders with their energy and pressing, and the latter felt he should have had a third-minute penalty when the ball popped up off Stophira Sunzu's arm.
The pressure was building on the Tunisia goal, but Mathlouthi was again on hand to deny Sunzu.
Kalaba remained the chief threat for Zambia, always willing to run in behind - and it was little surprise that it was he who crafted the breakthrough 14 minutes after the restart.
The skipper controlled a loose ball on the edge of the box drawing three defenders as he manoeuvred into a dangerous position before squaring for an unmarked Mayuka to smash home from six yards.
Mayuka could have doubled his tally three minutes later, but dragged his shot across goal and wide when played through one-on-one before then narrowly missing Kalaba's low cross with the goal gaping.
And those misses proved costly, as Tunisia levelled with their first meaningful attack of the half - Akaichi slotting home a flicked on corner at the back post, before Chikhaoui's last-gasp winner put them in control of Group B.
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