Introduction Bats are considered natural reservoirs for lyssaviruses. A total of 17 out of 19 known lyssaviruses circulate in bat populations. Lyssaviruses cause rabies in animals and humans. The transmission of lyssaviruses from European bats to terrestrial animals and humans is rare, but the risk of infection still exists even in developed countries. Slovakia is currently a rabies-free country. Objective The aim of the study was to assess the potential circulation of EBLV-1 in synanthropic bats present in human inhabited buildings, and to give an overview of human exposure to bats. Material and methods A passive serological survey targeted the prevalence of antibodies to bat lyssaviruses in synanthropic bats between 2009 – 2019. A total of 598 bats of the species Pipistrellus pipistrellus , Pipistrellus pygmaeus , Eptesicus serotinus, Nyctalus noctula and Vespertilio murinus were captured in buildings mainly in Eastern Slovakia, and examined by the rapid fluorescent focus inhibition test (RFFIT). Results Lyssavirus-specific antibodies were detected in 2 (0.3%) of the 598 examined bats. Additionally, brain tissues of bats found dead were examined using the standard fluorescent antibody test (FAT) with negative results. An overview of available data on human exposure to bats recorded in Slovakia from 2007 – 2019 is also included. Conclusions The study confirmed the presence of lyssavirus antibodies in synanthropic bats in Slovakia, suggesting the active circulation of bat lyssaviruses in bat populations exploiting human buildings. Although the seroprevalence was found to be extremely low, the results show that any case of human exposure to bats must be treated with caution in order to protect public health.