Enterococci are among the ubiquitously distributed opportunistic bacteria known for their involvement in healthcare-associated infections. Therefore, the aim of this study was to investigate the distribution of a broad spectrum of virulence determinants in the two major species of the genus Enterococcus in different contexts of origin and genetic lineages. We analyzed 322 isolates (166 E. faecium and 156 E. faecalis) from different sources [humans (hospitalized patients), coastal waters, and wild birds] in the state of Rio de Janeiro. The isolates were identified by MALDI-TOF MS, and a panel of 16 virulence genes was analyzed by PCR. BLAST tool was used to evaluate other virulence factors in WGS: seven genes of the enterococcal pathogenicity island, four pheromones genes, and the gelatinase (six genes) and cytolysin (eight genes) operons. Genetic lineages were determined by MLST. At least one gene from the panel examined by PCR was found in 97% of the strains. Acm was most abundant in E. faecium (86%). In E. faecalis, efaA and eep were found with a frequency of 96% each. Esp (49%; 55% in E. faecium and 42% in E. faecalis), ecbA (60%; 57% and 63%), and pilB (64%; 80% and 47%) were observed in both species. However, esp possessing E. faecium and ecbA possessing E. faecalis were mainly of human origin. Acm, pilA, sgrA, and scm were observed only in E. faecium, with higher rates in strains of human origin, and sgrA was not found in strains from coastal waters. Eep, efaA, ace, gelE, cob, aggA, cylA, and asa1 were observed at high rates only in E. faecalis, while cylA and aggA had lower rates in strains from wild birds and coastal waters. Regarding the co-circulation of genes in virulence profiles, 78 profiles were observed. The profiles esp-sgrA-pilA-scm-ecbA-acm-pilB in E. faecium and gelE-cylA-ace-efaA-eep-aggA-ecbA-pilB-cob in E. faecalis of human origin predominated, grouping 22% and 11% of strains, respectively. Gelatinase (gelE-sprE-fsrA-fsrB-fsrC-fsrD) and cytolysin (cylA-cylM-cylB-cyll-cylL-cylS-cylR1-cylR2) operons were only found in E. faecalis isolates and a small number of strains with incomplete operons. The same was observed for the pheromone genes cob, cpd, cad, and ccf, which were abundant. As for the pathogenicity island genes (esp-hyd-psaA-cylA-nuc1-cbh-gls24-like), only esp and psaA were detected in both species. The esp-psaA profile predominated E. faecium and hyd-psaA-cylA-nuc1-cbh in E. faecalis. Phylogenetic analyzes based on MLST revealed that most E. faecium strains of human origin belonged to lineages of ancient clonal complex CC17 (ST78, ST896, ST412, and ST943) and those of E. faecalis belonged to CC2 (ST6) and ST525, ST564, ST778. In addition, STs from more than one origin were identified: E. faecalis-ST40, ST21 and ST4; E. faecium-ST60, ST94, ST22 and ST25. Both species showed a large number of virulence profiles, with E. faecalis showing the greatest diversity. Profiles composed by the highest numbers of virulence determinants prevailed among the genetic lineages with the highest number of strains in both species. The presence of esp correlated with human hospital lineages in E. faecium and pilB with the frequency of strains in the E. faecalis and E. faecium lineages. The results indicate the widespread distribution of genes associated with virulence and underscore the importance of studies from a One Health perspective, which refers to the inseparable link connecting human, animal, and environmental health.