Background:Bartonellae are fastidious bacteria causing persistent bacteremia in humans and a wide variety of animals. In recent years there is an increasing interest in mammalian bartonelloses in general and in rodent bartonelloses in particular. To date, no studies investigating the presence of Bartonella spp. in rodents and ectoparasites from Nigeria were carried out.Methodology/Principal Findings:The aim of the current study was to investigate the presence of Bartonella spp. in commensal rodents and their ectoparasites in Nigeria. We report, for the first time, the molecular detection of Bartonella in 26% (46/177) of commensal rodents (Rattus rattus, R. norvegicus and Cricetomys gambianus) and 28% (9/32) of ectoparasite pools (Xenopsylla cheopis, Haemolaelaps spp., Ctenophthalmus spp., Hemimerus talpoides, and Rhipicephalus sanguineus) from Nigeria. Sequence analysis of the citrate synthase gene (gltA) revealed diversity of Bartonella spp. and genotypes in Nigerian rodents and their ectoparasites. Bartonella spp. identical or closely related to Bartonella elizabethae, Bartonella tribocorum and Bartonella grahamii were detected.Conclusions/Significance:High prevalence of infection with Bartonella spp. was detected in commensal rodents and ectoparasites from Nigeria. The Bartonella spp. identified were previously associated with human diseases highlighting their importance to public health. Further studies need to be conducted to determine whether the identified Bartonella species could be responsible for human cases of febrile illness in Nigeria.