The genus Bartonella comprises gram-negative bacilli that possess tropism for erythrocytes and endothelial cells in animals of the orders Rodentia, Lagomorpha, Carnivora, Artiodactyla, Eulipotyphla, and Chiroptera. Bacterial infection may be associated with lymphadenitis, endocarditis, bacillary angiomatosis, and peliosis hepatis. Thirteen species of Bartonella are recognized as zoonotic and bats are considered to be their potential reservoirs. This study aimed to analyze the occurrence of Bartonella spp. in livers of neotropical bats belonging to the families Molossidae, Phyllostomidae, and Vespertilionidae, classified into 21 genera from the Atlantic Forest biome in São Paulo. A total of 341 (n = 341) chiropterans samples were tested for the presence of citrate synthase (gltA) gene of Bartonella by partial amplification using polymerase chain reaction (PCR). Samples of two bats (0.6%) of the species Glossophaga soricina from the municipality of São Roque were tested positive for gltA gene. Phylogenetic analysis indicated that sequences clustered in a clade that was close to the Bartonella sp. detected in G. soricina, which was collected from the Cerrado biome in Tocantins, Brazil. Despite a low prevalence of the detected infection, results indicated that neotropical bats from the Atlantic Forest were potential hosts of Bartonella spp., which might be related to the maintenance of a wild enzootic cycle of the bacterium. Additional studies, particularly on bats of genus Glossophaga, are required to elucidate the dynamics of intraspecific relationships between etiologic agent-vector-hosts.