Mussels have a filter system enabling them to take up nutrients from the water, so a microbiological analysis of these bivalve mollusks can show the contamination levels of their surrounding aquatic environment. The present work aimed to isolate Vibrio species from two hundred samples of mussels (Perna perna) incrusted on rocks of the Santana Archipelago and from longline mariculture in Ilha Grande Bay in Angra dos Reis and from Arraial do Cabo, all of which are in Rio de Janeiro state, Brazil. A total of 209 Vibrio were isolated. The most prevalent species was Vibrio parahaemolyticus (44.66%) followed by Vibrio alginolyticus (19.62%) and Vibrio vulnificus (12.44%). All 209 Vibrio isolates tested positive for the RNA polymerase alpha gene (rpoA). The tlh gene (thermolabile hemolysin), a genetic marker for V. parahaemolyticus, and vvhA (cytolysin hemolysin) of V. vulnificus were detected in 85 and 26 isolates, respectively. The MALDI-TOF MS proteomic technique was used to confirm the identification of the 41 V. alginolyticus isolates. Our most important finding was the detection of the tdh virulence gene in 68.20% (58/85) of V. parahaemolyticus environmental strains. Besides the circulation of the virulence gene, the spread of antimicrobial resistance was evaluated and 91.3% (191/209) of the isolates showed resistance to ampicillin, 23.9% (50/209) to ciprofloxacin, 18.6% (39/209) to nitrofurantoin, 5.7% (12/209) to tetracycline, 4.3% (9/209) to pefloxacin and 3.3% (7/209) to chloramphenicol. These findings indicate that environmental isolates can act as reservoirs of virulence and antibiotic resistance genes.