The black-eared opossum (Didelphis aurita) is a South American synanthropic marsupial. The presence of opossums in domestic spaces is relevant in the One-Health context since they are hosts of pathogens and ectoparasites that may affect the health of domestic animals and humans. In this study, we aim to determine the occurrence of hemoplasmas and selected tick-borne pathogens in free-ranging black-eared opossums, along with their molecular characterization, hematological and biochemical evaluation and factors associated with infection, in the municipality of Viçosa, State of Minas Gerais, southeastern Brazil. Thirty black-eared opossums were trapped between March 2021 and June 2022. Ectoparasites were collected. Hematological and biochemical analyses were performed. DNA from EDTA-blood samples were analyzed by PCR and qPCR assays. By molecular analyses, ‘Candidatus Mycoplasma haemoalbiventris’ was the most prevalent hemoparasite (73.3%), followed by Hepatozoon sp. (22.2%). Significant differences were observed in the number of platelets, and in the concentration of protein and globulins in the animals infected by ‘Ca. M. haemoalbiventris’ when compared with the negative group. This is the first report of ‘Ca. M. haemoalbiventris’ infection in D. aurita.