Emergence of different pathogenic strains has changed the status of Escherichia coli from a non-pathogenic feacal indicator bacterium to a leading global pathogen. Seafood is highly susceptible to contamination by E. coli at various stages from harvest to consumption. In this study, E. coli was isolated from the seafood collected from different landing centers and markets in Mumbai, India. The isolates were identified and confirmed as E. coli biochemically and by Polymerase Chain Reaction (PCR). The isolates were further characterized by screening for different virulence genes characteristic of Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli (ETEC), Enterohemorrhagic E. coli (EHEC), Enteroinvasive E. coli (EIEC), Enteroaggregative E. coli (EAEC) and Diffusely adherent E. coli (DAEC). All the samples harbored E. coli suggesting widespread fecal contamination of seafood. Out of 400 isolates screened from 20 seafood samples, 224 (56%) were confirmed as E. coli. Of these 224 E. coli isolates, 122 (54.5%) isolates were from fish and 102 (45.5%) were from shellfish. Comparative analysis of isolation media revealed that MacConkey agar, Eosin Methylene Blue Agar and Sorbitol MacConkey Agar allowed better isolation of E. coli from seafood compared to HiChrome agar. Virulence genes such as eaeA, bfpA, stx1, stx2, hlyA, st, lt and aggR were detected in seafood isolates of E. coli by PCR, suggesting the occurrence of the different pathotypes of E. coli in seafood. The result of this study strongly suggest the presence of pathogenic E. coli in seafood. There is a need to understand the routes of contamination of seafood and the health implications due to the presence of different pathotypes of E. coli in seafood. Suitable control measures are urgently needed to prevent seafood contamination with pathogenic E. coli and ensure the safety of seafood for consumption.