Leptospirosis is a bacterial zoonotic infection of worldwide occurrence. Bats, like other mammalian reservoirs, may be long-term carriers that maintain endemicity of infection and shed viable leptospires in urine. Direct and/or indirect contact with these Leptospira shedders is the main risk factor as regards public health concern. However, knowledge about bat leptospirosis in the Palearctic Region, and in Europe in particular, is poor. We collected urine from 176 specimens of 11 bat species in the Czech Republic, Poland, Republic of Armenia and the Altai Region of Russia between 2014 and 2019. We extracted DNA from the urine samples to detect Leptospira spp. shedders using PCR amplification of the 16S rRNA and LipL32 genes. Four bat species (Barbastella barbastellus n = 1, Myotis bechsteinii n = 1, Myotis myotis n = 24 and Myotis nattereri n = 1) tested positive for Leptospira spp., with detected amplicons showing 100% genetic identity with pathogenic Leptospira interrogans. The site- and species-specific prevalence range was 0%–24.1% and 0%–20%, respectively. All bats sampled in the Republic of Armenia and Russia were negative. Given the circulation of pathogenic leptospires in strictly protected Palearctic bat species and their populations, non-invasive and non-lethal sampling of urine for molecular Leptospira spp. detection is recommended as a suitable surveillance and monitoring strategy. Moreover, our results should raise awareness of this potential disease risk among health professionals, veterinarians, chiropterologists and wildlife rescue workers handling bats, as well as speleologists and persons cleaning premises following bat infestation.