Background:Vibrio choleraeis an epitome of non-invasive organisms, which only affects the small intestine via the liberation of enterotoxin.The plasmid profile and beta lactamase production in ampicillin-resistant Vibrio species isolated from seafoods collected from coastal area of Ondo State, Nigeria were investigated. Methods: One hundred and six (106) samples of seafoods were collected in February, 2020 and January, 2021. The isolation and identification of Vibrio species, antibiotic sensitivity pattern, plasmid analysis and beta-lactamase detection activity were conducted via pour plate, Kirby-Bauer disc diffusion, alkaline lysis, neutralization, electrophoresis methods, use of 10 % sodium dodecyl sulphate (SDS) and double disk synergistic test (DDST) respectively. Results: Pathogenic bacteria such as Bacillus subtilis, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, and Vibrio cholerae were isolated from 10 crabs, 54 fishes, and 42 shrimps. Findings also revealed that 66% of Vibrio cholerae that showed multiple antibiotic resistance tested positive to beta lactamase production test. The result of plasmid analysis indicated that they were more susceptible to antibiotics after plasmid curing of the microorganisms. Conclusion: The resistance of Vibrio cholerae in this investigation was found to be plasmid-mediated consequent to its loss after curing. The detection of multidrug resistance, ESBLs-producing Vibrio organisms, suggests risks to public health.The risk of seafood-borne infections can be reduced by decrease in pollution levels, ensuring appropriate cooking temperatures, and prevention of cross-contamination during harvesting, handling and processing.