Bats comprise one quarter of the world's mammal species. In Europe, three nidicolous Ixodes tick species, I. vespertilionis, I. simplex and I. ariadnae are specifically associated with cave-dwelling bats, but their role as potential vectors of zoonotic agents is unknown. In this study, we used PCR-based methods to provide the first evidence of Borrelia burgdorferi sensu lato (s.l.) infections in the three bat-associated tick species collected from ten bat species sampled in Poland and Romania. B. burgdorferi s.l. was detected in 24% (64/266) of tick samples, and 40.3% (60/149) of the bats carried infected chiropterophilic ticks. In Poland, the B. burgdorferi s.l. infection prevelance of I. ariadnae ticks parasitizing Myotis species was four times higher compared to the I. vespertilionis ticks derived from Rhinolophus hipposideros bats (44.4% vs.10%, respectively). The observed differences in infection prevalence could be explained by differences in reservoir potential between bat species. Bats from the genus Myotis and Miniopterus schreibersii carried more infected ticks than R. hipposideros regardless of the tick species. Analysis of the flaB gene sequences revealed seven species from the B. burgdorferi s.l. complex (B. afzelii, B. carolinensis, B. garinii, B. lanei, B. spielmanii, B. burgdorferi s.s., and B. valaisiana), of which five are considered as human pathogens. This large diversity of Borrelia species may reflect differences in susceptibility of chiropteran hosts and/or the tick vectors. Generally, mammal-associated B. burgdorferi s.l. species were more common than bird-associated species. Our study provides evidence for new enzootic transmission cycles of B. burgdorferi s.l. spirochetes involving nidicolous Ixodes tick species and cave-dwelling bats. © 2019 Elsevier GmbH