Leptospirosis is a worldwide zoonosis which has been recently recognized as a paradigm to the One Health approach due to the interface of human-animalenvironment observed in the transmission cycles. A total of 40 opossums identified as Didelphis aurita were captured at the Campus FIOCRUZ within the Atlantic Forest, Rio de Janeiro, Brazil to evaluate their possible role as carriers of Leptospira spp. The 40 serum samples were submitted to the microagglutination test using a panel of 19 reference strains. Kidney fragments of 13 animals out of 40 were used to perform PCR and standard procedures to isolate leptospires in culture. The percentage of positive sera was 10% (4 out of 40). The PCR showed 4 positive kidney samples out of 13 (31%). Two strains were isolated in culture medium (15.4%). Multilocus sequence typing (MLST) analysis of both isolates did not show a 100% match with any other sequence types deposited at the database used (http://pubmlst.org/leptospira/). The closest match of one isolate was with ST 177 represented by one strain of L. santarosai and the closest match of the other isolate was with STs 166 and 171 represented by strains of L. noguchii. It is the first report indicating the potential of opossums Didelphis aurita as a carrier of Leptospira spp.