A small outbreak of gastroenteritis in 2011 in Apalachicola Bay, FL was attributed to consumption of raw oysters carrying Vibrio cholerae serotype O75. To better understand possible health risks, V. cholerae was surveyed in oysters, fish and seawater, and results were compared to data for Vibrio vulnificus and Vibrio parahaemolyticus.Enrichment protocols were used to compare prevalence of V. cholerae (0, 48, 50%), V. vulnificus (89, 97, 100%) and V. parahaemolyticus (83, 83, 100%) in fish, seawater and oysters respectively. Compared to other species, Most probable number results indicated significantly (P < 0·001) lower abundance of V. cholerae, which was also detected more frequently at lower salinity, near-shore sites; other species were more widely distributed throughout the bay. Genes for expression (ctxA, ctxB) and acquisition (tcpA) of cholera toxin were absent in all strains by PCR, which was confirmed by whole genome sequencing; however, other putative virulence genes (toxR, rtxA, hlyA, opmU) were common. Multi-locus sequence typing revealed 78% of isolates were genetically closer to V. cholerae O75 lineage or other non-O1 serogroups than to O1 or O139 serogroups. Resistance to amoxicillin, kanamycin, streptomycin, amikacin, tetracycline and cephalothin, as well as multidrug resistance, was noted.Results indicated minimal human health risk posed by V. cholerae, as all isolates recovered from Apalachicola Bay did not have the genetic capacity to produce cholera toxin. Vibrio cholerae was less prevalent and abundant relative to other pathogenic Vibrio species.These studies provide important baseline observations for V. cholerae virulence potential regarding: (i) genetic relatedness to V. cholerae O75, (ii) antibiotic resistance and (iii) prevalence of multiple virulence genes. These data will serve as a biomonitoring tool to better understand ecosystem status and management if bacterial densities and virulence potential are altered by environmental and climatic changes over time.