The competition started on 16 September 2009 with the first round and ended with the final held in spring 2010.
The defending champions were Sheriff, who won their sixth cup final last season, and defended their title this season.
This round involved 16 teams, including four from the Moldovan National Division: CSCA-Rapid, Academia, Viitorul Orhei and Sfîntul Gheorghe.
This round featured the eight winners from the previous round as well as the eight remaining clubs from the Moldovan National Division.
The matches were played on 30 September 2009 except for the match between Sheriff and CSCA-Rapid, which took place on 22 November 2009.