While the lesser horseshoe bat (Rhinolophus hipposideros) was common in most western and Central Europe, this species endured a dramatic decline in the 1950s–1970s. The causes are thought to comprise the extensive spread of chemical pollutants affecting both bat and prey populations, changes in landscape composition and structure leading to population fragmentation or roost deterioration, and infectious diseases. While lesser horseshoe bat populations recently increased after nearly thirty years of decline in the Franche-Comté region (eastern France), it is unclear whether bats are currently confronted with causes that may have contributed to former demographic disturbances. Although stressors are generally studied separately, we simultaneously measured several variables directly or indirectly reflecting the supposed causes of bat decline: availability of woodland habitats around bat roosts, genetic differentiation amongst colonies, and exposure to chemical pollutants and to a protozoan parasite, Eimeria hessei. Twenty maternity colonies were sampled using a non-invasive approach based on droppings. Maternity roosts were located in buildings mainly surrounded by woodland and shrubland (48% of a 2500 m radius