The Conservative Civic Democratic Party (ODS) won the election with 10 seats.
The Communist Party lost its last seat in the Senate when Václav Homolka failed to be re-elected, meaning the party would be without representation in the Senate for the first time in the history of Czech Republic.
The election was considered the first major win for the opposition to Andrej Babiš' Cabinet.
Members of the Senate are elected in single-member constituencies using the two-round system.
Babiš said that ANO was offering many highly qualified candidates, including Ivan Pilný and Eva Syková.