Background: Lice are blood-sucking insects that are of medical and veterinary signi cance as parasites and vectors for various infectious agents. More than half of described blood-sucking lice species are found on rodents. Rodents are important hosts of several Bartonella and Rickettsia species and some of these pathogens are characterised as human pathogens in Europe. Rodent ectoparasites, such as eas and ticks, are important vectors of Bartonella spp. and Rickettsia spp., but knowledge about the presence of these bacteria in lice is limited. The aim of this study was to determine the prevalence of Bartonella and Rickettsia bacteria in lice collected from rodents in Slovakia. Methods: The ectoparasites were collected from small rodents captured from 2010 to 2015 at four different sites in eastern Slovakia. The presence of Bartonella and Rickettsia pathogens in lice samples was screened by real-time PCR, targeting ssrA and gltA genes respectively. The molecular characterisation of the Bartonella strains was based on sequence analysis of partial rpoB and ITS genes, and of the Rickettsia species on sequence analysis of the gltA gene. Results: A total of 1074 lice of seven species were collected from six rodent species in Slovakia from 2010 to 2015. Bartonella DNA was detected in three species of lice Hoplopleura a nis (collected from Apodemus agrarius, A. avicollis and Myodes glareolus), Polyplax serrata (from A. agrarius) and Hoplopleura sp. (from A. avicollis). Sequence analysis revealed that the Bartonella strains belonged to the B. coopersplainsensis, B. tribocorum and B. taylorii genogroups. Rickettsia DNR was detected in H. a nis and P. serrata lice collected from A. agrarius. Sequence analysis revealed the presence of two Rickettsia species: R. helvetica and Rickettsia sp. Conclusions: To the best of the authors' knowledge, this is the rst report on the occurrence and diversity of Bartonella spp. and Rickettsia spp. in lice collected from small rodents in Europe. This study is also the rst to detect B. coopersplainsensis in Slovakia.