In order to define appropriate conservation measures in northern Tunisia we surveyed the bat fauna of Mastouta-Bishshouk region in both roosting sites and foraging areas. A total of 11 species was recorded. We found only three occupied roosts including a maternity colony of Rhinolophus ferrumequinum, Miniopterus schreibersii and Myotis cappacinii in a train abandoned tunnel. A tunnel of water channel hosted Rhinolophus mehelyi, M. schreibersii and M. punicus. Echolocation calls were recorded at a bridge over Beja Wadi, at an artificial basin, and in crop fields. Bat activity was the highest at the bridge in late March, and null in the harvested crop fields in late August. Pipistrellus kuhlii was the most active bat species in the three sites, Pipistrellus pipistrellus was the second most active species, followed by Eptesicus isabellinus. Plecotus gaisleri and Myotis punicus were detected only once at the bridge and the basin respectively. This bat assemblage is representative of the bat fauna of northern Tunisia. Roost and foraging area surveys strongly benefit acoustic recording, even in winter when some bats remain active. The rare local underground roosts should be protected from human disturbance and the water quality of Majerda Wadi should be improved.