The seasonal activity of the Isabelline serotine bat Eptesicus isabellinus was studied in eight habitats of the Bou Hedma National Park, central Tunisia. From June 2010 to June 2011 two techniques, mist-netting and echolocation call recording, were implemented. This bat species was captured throughout the year but only at water bodies and particularly at Nouh basin. Captures peaked in late spring — early summer, and declined in winter. Pregnant females were captured from April to June, and lactating females in May and June. Echolocation calls were recorded throughout the year, but not every month in all habitats. Most activity was reported at water bodies and street lamps. Activity peaked in late spring and early autumn, and declined during winter period. Terminal buzzes were mainly detected at basins, peaking in late spring — early summer and early autumn. Our study provided further insights on the importance of water bodies which should draw particular attention in the management plan of the national park and in the whole country.