Background: Feline Bartonella can be transmitted to humans through cat scratches or bites, and between cats by Ctenocephalides felis flea. Methods: The occurrence of Bartonella DNA was assessed in ectoparasites and their cat hosts living in shelters based on the ITS region and gltA gene. Results: Bartonella DNA was detected in 47.8% of cat blood samples, in 18.3% of C. felis fleas,13.3% of flea eggs pools and 12.5% of lice pools. B.henselae and B. clarridgeiae DNA were detected in cat fleas, while B. henselae, B. clarridgeiae, and B. koehlerae in blood samples from bacteremic cats. Cats infested by positive ectoparasites showed approximately twice the chance of being infected. Conclusions: Our results indicate shelter cats have a high prevalence of Bartonella species known to be human pathogens and highlight the importance of controlling their infestation by ectoparasites to avoid cat and human infection.