This is the first brief communication of clinical mastitis caused by Staphylococcus aureus occurring in a white-eared opossum (Didelphis albiventris) captured in a remnant of the Atlantic Forest in northeast Brazil. The bacterium was cultured from a milk sample by using sheep blood and Levine agars. Phenotyping of S aureus was performed with coagulase biochemical tests, DNase agar, mannitol agar, and anaerobic glucose and mannitol fermentation.