In captive environments, mammals are frequently exposed to various parasitic protozoa and other vector-borne pathogens that can impact both animal health and public health. Monitoring these pathogens is essential for animal welfare and zoonotic disease control. This study aimed to investigate the prevalence of parasitic protozoa and other vector-borne pathogens in captive mammals through molecular detection methods at the Belo Horizonte Zoo, Brazil. Between November 2021 and March 2023, whole blood samples were collected from 40 mammals. Molecular analyses identified piroplasms, Leishmania spp., granulocytic/platelet Anaplasma/Ehrlichia spp., monocytic Ehrlichia spp., Bartonella spp. and hemotropic Mycoplasma spp. with a 72.5% positivity rate. Piroplasms were found in 22.5% (two Pantanal cats, two gorillas, one white rhinoceros, one spider monkey, one jaguar, one tufted capuchin and one hippo) and Leishmania spp. in 12.9% (four maned wolves). Granulocytic/platelet Anaplasma/Ehrlichia spp. were found in 12.5% of the samples (one gorilla and four maned wolves), Ehrlichia canis in 2.5% of the animals (one maned wolf), Bartonella spp. in 42.5% (six howler monkeys, two maned wolves, one gorilla, one white rhino, one southern tamandua, one common woolly monkey, one tufted capuchin, one brown brocket deer, one agouti, one cougar and one hippo), hemotropic Mycoplasma spp. in 17.5% (one gorilla, one maned wolf, one white rhino, one howler monkey, two common woolly monkeys and one European fallow deer). Five Artiodactyla members tested negative for A. marginale. Coinfections occurred in 34.5% of the positive samples. Sequencing revealed that Theileria spp. and Cytauxzoon spp. are closely related to Theileria bicornis and Cytauxzoon felis; Ehrlichia canis and Bartonella spp. are closely related to B. clarridgeiae and B. henselae; and hemotropic Mycoplasma spp. are closely related to Candidatus Mycoplasma haemominutum. Our results showed a high occurrence of vector-borne pathogens in captive animals, including zoonotic species, which may pose a risk to animal and human public health.