Asymptomatic cats often harbor pathogens, some of which have not been largely investigated in feline populations. The aim of this study was to evaluate the occurrence of antibodies against Rickettsia conorii, Rickettsia felis, Rickettsia typhi, Neospora caninum, Bartonella henselae and Toxoplasma gondii in cats from Tuscany. Ninety-five blood serum samples, previously collected, were analyzed by indirect immunofluorescence assay. Fifty-six (58.94%) cats had antibodies to at least one investigated pathogen: 28 (29.47%) cats were positive for B. henselae, 17 (17.89%) for R. felis, 14 (14.73%) for R. conorii, 14 (14.73%) for T. gondii, 2 (2.1%) for N. caninum. No cats were positive for R. typhi. Positive reactions to two or more pathogens were detected in 18 (18.94%) cats. The occurrence of antibodies against these microorganisms suggests that cats, even though asymptomatic, may be infected by pathogens, often zoonotic, and thus may be a source of infections for other animals and humans.