Abstract We report the molecular detection of Borrelia afzelii (11%) and Bartonella spp. (56%) in 447 bank voles trapped in a suburban forest in France. Adult voles were infected by significantly more Borrelia afzelii than juveniles (p < 0.001), whereas no significant difference was detected in the prevalence of Bartonella spp. between young and adult individuals (p = 0.914). Six percent of the animals were co-infected by both bacteria. Analysis of the bank vole carrier status for either pathogen indicated that co-infections occur randomly (p = 0.94, CI95 = [0.53; 1.47]). Sequence analysis revealed that bank voles were infected by a single genotype of Borrelia afzelii and by 32 different Bartonella spp. genotypes, related to three known species specific to rodents (B. taylorii, B. grahamii and B. doshiae) and also two as yet unidentified Bartonella species. Our findings confirm that rodents harbor high levels of potential human pathogens; therefore, widespread surveillance should be undertaken in areas where humans may encounter rodents.