Recently, tick and flea-borne pathogens have been detected in wild carnivores maintained in captivity inBrazilian zoos. Since free-roaming cats are frequently found in Brazilian zoos, they could act as reservoirsfor arthropod-borne pathogens, which could be transmitted to endangered wild carnivores maintainedin captivity in these institutions. On the other hand, stray cats in zoos may play a role as sentinels topathogens that circulate among wild animals in captivity. The present work aimed to detect the presenceof Anaplasmataceae agents, hemoplasmas, Bartonella species, piroplasmas, and Hepatozoon sp. DNA inblood samples of 37 free-roaming cats in a Brazilian zoo. Three (8%) cats were positive for Anaplasma spp.closed related to Anaplasma phagocytophilum; 12 (32%) cats were positive for hemoplasmas [two (5%)for Mycoplasma haemofelis, five (13.5%) for Candidatus Mycoplasma haemominutum, and five (13.5%) forCandidatus Mycoplasma turicensis]; 11 (30%) were positive for Bartonella spp., six (16%) were positiveBabesia vogeli and one (3%) for Theileria sp. Coinfection with multiple arthropod-borne agentes wasobserved in sampled cats. None of sampled cats were positive for Ehrlichia spp., Cytauxzoon spp., orHepatozoon spp. in PCR. This is the first molecular detection of Babesia vogeli and Theileria sp. in domesticcats in Brazil. The control of the population of free-roaming cats in these conservation institutions ismuch needed aiming to prevent the potential transmission to endangered wild animals maintained incaptivity, such as wild neotropical wild felids, as well as to human beings visiting zoos