Five days ahead of the Africa Cup of Nations, Tunisia announced Tuesday that a second player in their squad, Seifeddine Jaziri, had tested positive for Covid-19.
On Monday, the Tunisian football federation revealed that midfielder Youssef Msakni had also tested positive.
Jaziri was top scorer at the Arab Cup in Qatar in December, scoring four goals as Tunisia reached the final where they lost 2-0 to Algeria.
Tunisia are not permitted to replace either player as squads were expanded from the usual 23 to 28 to cover potential problems with coronavirus.
Tunisia have won the African title only once in their history, when they hosted the competition in 2004. They reached the semi-finals of the last edition in 2019.
The Eagles of Carthage begin their Group F campaign against Mali on January 12 before going on to face Mauritania and Gambia.