The genus Vibrio comprises pathogens ubiquitous to marine environments. This study evaluated the cultivable Vibrio community in the Guanabara Bay (GB), a recreational, yet heavily polluted estuary in Rio de Janeiro, Brazil. Over one year, 66 water samples from three locations along a pollution gradient were investigated. Isolates were identified by MALDI-TOF mass spectrometry, revealing 20 Vibrio species, including several potential pathogens. Antimicrobial susceptibility testing confirmed resistance to aminoglycosides, beta-lactams (including carbapenems and third-generation cephalosporins), fluoroquinolones, sulfonamides, and tetracyclines. Four strains were producers of extended-spectrum beta-lactamases (ESBL), all of which carried beta-lactam and heavy metal resistance genes. The toxR gene was detected in all V. parahaemolyticus strains, although none carried the tdh or trh genes. Higher bacterial isolation rates occurred in months marked by higher water temperatures, lower salinities, and lower phosphorus and nitrogen concentrations. The presence of non-susceptible Vibrio spp. was related to indicators of eutrophication and sewage inflow. DNA fingerprinting analyses revealed that V. harveyi and V. parahaemolyticus strains non-susceptible to antimicrobials might persist in these waters throughout the year. Our findings indicate the presence of antimicrobial-resistant and potentially pathogenic Vibrio spp. in a recreational environment, raising concerns about the possible risks of human exposure to these waters.