Enterococci are ubiquitous microorganisms found as part of the normal intestinal microbiota of many animals such as the free-ranging Eurasian otter (Lutra lutra Linnaeus, 1758). In this work, twenty-nine enterococci isolated from fecal samples of Eurasian otters free-living in reservoirs and associated river stretches in South Portugal were identified and typed by conventional/molecular methods and screened for virulence factors and antibiotic resistance. Identification allocated the isolates to the species Enterococcus faecalis (19), E. faecium (9) and E. durans (1) and PCR-fingerprinting revealed their high genomic diversity. Regarding virulence factors, three isolates produced cytolysin and six were gelatinase-positive. Genes ace and acm were detected in five enterococci each, ebpABC in seventeen, gelE in fourteen and cylA in three. All isolates showed resistance patterns and antibiotic resistance genes tet(M) and pbp5 were detected in seventeen isolates each, whereas vanB and vanD were identified in thirteen and five, respectively, being most van-harboring isolates members of E. faecium. The aac(6')-Ie-aph (2 `') gene, encoding for gentamicin resistance, was observed in all gentamicin-resistant enterococci. Since all isolates harbor virulence and/or antibiotic resistance traits, the role of free-living Eurasian otters in the dissemination of virulent/resistant enterococci among other animals sharing the same ecological niche cannot be disregarded, as well as the health risk they may represent for humans directly interacting with them or their habitat. (C) 2013 Elsevier B.V. All rights reserved.