Cats are considered main reservoir of Bartonella henselae, which is transmitted to other cats especially through Ctenocephalides felis fleas, and to humans through scratching and biting. Serra da Tiririca State Park (PESET) is an Atlantic Forest area that shelters a wide variety of endemic fauna. Recently, the park has been suffering due to irregular housing construction and domestic animal population that interacts with humans and wildlife. Given that surveillance policies for animals are part of the global Strategic Framework for One Health, the aim of this study was to detect Bartonella spp. DNA in cats and dogs, evaluating laboratory changes and associated factors. Blood samples of 124 dogs and 89 cats were collected for hematology and serum chemistry analysis. DNA was extracted and tested by conventional polymerase chain reaction (PCR) targeting a fragment of the citrate synthase (gltA) gene of Bartonella spp. with specific primers. Positive samples were sequenced to identify species. Bartonella henselae and B. clarridgeiae were detected in 24.7% of cats, being, for our knowledge, the first report of B. clarridgeiae in cats from Rio de Janeiro, Brazil. None of the samples obtained from dogs tested positive in the PCR assays. No statistical significance was observed in physical and laboratory exams. We suggest that cats that inhabit PESET can be considered sources of Bartonella sp. for other cats and humans. We highlight that infected cats did not present clinical or laboratory alterations. We alert for the need of care measures, avoiding scratch and bite, particularly in immunocompromised people.