Recent studies suggest that the damage caused by roads on bats may significantly affect bat populations. However, despite bats being one of the most threatened groups of European vertebrates, scientific information is still lacking to effectively mitigate this effect. We daily surveyed roadkilled bats in 52 km of roads with different traffic volume in southern Portugal, between March and October 2009. During the same period we also monitored bat activity in the study area. We found 154 roadkilled bats of 11 species. Pipistrellus kuhlii and P. pygmaeus represent 64 % of total specimens collected. We also found threatened and poorly known species like Barbastella barbastellus (2), Rhinolophus ferrumequinum (1), R. hipposideros (5), Miniopterus shreibersii (1) and Nyctalus leisleri (1). A peak of mortality occurred in late summer with 67% of bats being killed between 15 of July and 15 of September. The males were killed more often than females, except in June when this pattern was inverted. The mortality of young was recorded only from June onwards and was a small percentage of total kills (approx. 16%). The relationship between bat fatalities and different types of explanatory variables (landscape, road characteristics and bat ecology) was assessed by generalized linear model (GLM) and variance partition techniques. Overall, landscape features explained about 37% variance (pure+joint effects) in bat mortality and were the most important of the three variable sets analyzed. Nevertheless, bat activity, traffic and the proximity to known roosts also had a significant influence on mortality. The results have shown an increase in mortality in road sectors crossing high quality habitat (forests, streams and pond vicinity); near bat roosts and core activity areas; and on high traffic roads. The importance of these findings for the management and implementation of effective mitigation measures of bat roadkills are discussed in this communication.