Vibrio cholerae is the etiologic agent of cholera. It is an autochthonous inhabitant of all aquatic environments. The virulence of V. cholerae is maintained by the CTX genetic element and tcpA gene. In the present investigation, environmental strains of V. cholerae isolated from different aquatic biotopes in Kerala were identified and serotyped. The antibiotic resistance pattern and presence of virulence and regulatory genes were examined. We found the presence of toxigenic non-O1/non-O139 strains harboring the CTX genetic element, heat-stable enterotoxin, rtxA gene, El Tor hemolysin, and Vibrio pathogenicity island (VPI). The strains also produced the cholera toxin (CT) as determined by monosialoganglioside enzyme-linked immunosorbent assay. A few strains belonging to the O1 serogroup but lacking the CTX genetic element were also observed. The majority of the environmental strains belonged to non-O1/non-O139 serogroup with many possessing toxR, ompU, heat-stable enterotoxin, and rtxA gene. The toxigenic non-O1/non-O139 strains exhibited resistance to trimethoprim, ampicillin, and polymixin B and intermediate resistance to co-trimoxazole. However, all other environmental strains were found resistant to ampicillin and polymixin B. Our findings demonstrate that the virulence genes are dispersed among the environmental strains of V. cholerae and a complex aquatic environment can give rise to pathogenic V. cholerae.