The purpose of this study was to determine the prevalence of Bartonella species and Rickettsia species DNA in the blood, oral cavity, skin and claw beds of feral cats without evidence of skin disease that were housed in Alabama (n = 24), Florida (n = 27) and Colorado (n = 32). Samples were assessed by use of polymerase chain reaction assays. The Bartonella species IgG prevalence was also determined. While Bartonella species DNA was not amplified from any sample from Colorado cats, it was commonly amplified from blood (56.9%), skin (31.4%), claws (17.6%) and gingiva (17.6%) of the 51 cats housed in Alabama and Florida. All 10 flea groups assessed in this study were infected with a Bartonella species or R. felis. Bartonella species IgG titres did not accurately predict bacteraemia (positive predictive value = 57.1%; negative predictive value = 82.1%). Bartonella species DNA was amplified from blood of cats with and without C. felis. Rickettsia felis DNA was only detected in or on the skin of one cat and the gingiva of an additional cat. It was concluded that cats can be an occupational health risk for veterinarians, particularly in areas with high prevalence of Ctenocephalides felis. Further study is required to determine whether Bartonella species or Rickettsia species infections of cats are associated with dermatological disease. The combination of Bartonella species serological test results with Bartonella species PCR or culture is likely to give the most accurate information concerning the current infection status of individual cats.