Background: The survival of pathogens in biofilms poses a threat to food safety. The aim of this study was to determine prevalence, antimicrobial resistance, and molecular characterization of Escherichia coli strains. Methods: Swab samples (n=144) were collected from biofilm formed on food contact surfaces in seafood pre-processing plant in India. E. coli was isolated and identified using uid A gene by Polymerase Chain Reaction (PCR). The risk assessment of the isolates was carried out in terms of their drug resistance and the presence of virulence genes. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method. Phylogenetic grouping was done by quadruplex PCR. Molecular typing of the strains was performed by Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR). Data were statistically analyzed using SPSS version 22. Results: Enteropathogenic E. coli (EPEC) strains were the most prevalent serotype. Multiplex PCR analysis revealed the presence of shiga toxin genes (stx1, stx2), intimin (eae), and enterohemolysin genes (hlyA). Shiga toxin gene stx2 showed the highest prevalence (83.33%). Among various phylogroups, B1 (45.56%) and B2 (30%) were the most prevalent phylogroups. Resistance to ampicillin (85.56%), piperacillin (84.44%), and cefpodoxime (85.56%) was widespread among the E. coli strains. Conclusion: The presence of genetically heterogeneous multi drug resistant E. coli strains with virulence potential showed a high risk in the seafood industry.