During the last decades, the important role of bats in ecosystems and the threats to many bat species were increasingly recognized, and bats were integrated into several conservation acts. Protected areas like national parks may play a major role in bat conservation if species requirements are included in management plans. With its high bat diversity and many unexplored off-shore islands, Sardinia has a high potential for the protection of European bats and for research on their ecology. We conducted the first survey of the bat fauna on the Sardinian off-shore island and National Park Asinara, a formerly inhabited island. We applied a multi-method approach over two years to create a proper species inventory and to obtain data on the use of man-made structures by the bats. We recorded bat calls at different sites in summer and winter, captured bats via mist-netting, and performed roost surveys of man-made structures. Ten species were identified, three of them are highly protected, namely Rhinolophus hipposideros, R. ferrumequinum and Miniopterus schreibersii. Thirty-nine day roosts and 14 night roosts of, in total, five species were found in man-made structures. Many structures provided day and night roosts for different bat species. Although forest and water bodies are limited, and intensive grazing has created several open areas on the island, a remarkably high bat diversity developed, making Asinara an important offshore retreat area for bats in Italy. Our findings indicate an essential role of the abandoned buildings, and artificial ponds, of the island for bat conservation. The results of this study provide a basis for future bat conservation measures on Asinara Island.