Bat fleas (Insecta: Siphonaptera: Ischnopsyllidae) are highly host-specific ectoparasites distributed worldwide (except Antarctica). The identification of flea species and the detection of flea-borne pathogens plays a significant role not only in veterinary and public health, but also in providing insights into the ecology, behaviour, and geographical distribution of fleas and flea-associated pathogens. The present study aimed to conduct a morphological and molecular identification of fleas collected from Lithuanian bats, as well as to screen for the presence of vector-borne pathogens. A total of 58 flea specimens were collected from bats in Lithuania. Based on morphological analysis, seven flea species were identified (Ischnopsyllus variabilis, I. octactenus, I. simplex, I. elongatus, I. hexactenus, Nycteridopsylla pentactena, and N. eusarca) and screened for the presence of Anaplasma spp., Borrelia spp., Babesia spp., Bartonella spp., Rickettsia spp., and Mycoplasma spp. Molecular analysis of fleas’ COI and 18S rRNA genes showed that the flea species collected from bats are closely genetically related. Bartonella DNA was detected in bat fleas (41.4%). No DNA of Anaplasma, Borrelia, Babesia, Rickettsia, and Mycoplasma was amplified in the tested samples. The Wolbachia sp. was accidentally found in I. elongatus. The phylogenetic analysis of four Bartonella genes (16S-23S rRNA ITS, gltA, rpoB, and groEL) revealed specificity to bats or bats’ ectoparasites. This is the first report about bat flea species and the pathogens in bat fleas in Lithuania. New host records for bat fleas were also reported. This study extends the knowledge about bat fleas and their role as vectors of different pathogens.