Seasonal prevalence of faecal coliforms in Indian black clam (Villorita cyprinoides), sediment and harvesting waters from shellfish harvesting areas along Cochin estuary was evaluated. Enumeration was done by 5 tube most probable number (MPN) method. High prevalence of faecal coliforms was observed in shellfish, sediment and harvesting waters during monsoon and post-monsoon seasons compared to pre-monsoon season. Escherichia coli was isolated and identified by biochemical methods and further confirmed by PCR based detection of uid A gene. A total 125 E. coli strains were isolated; out of which 99 (79.2%) could be serotyped and comprised of 17 diverse serotypes. Approximately 89.06% of the serotyped strains belonged to 5 different pathogenic serotypes. Enterotoxigenic strains were the most prevalent, followed by enterohaemorrhagic, uropathogenic, enteropathogenic and enteroinvasive strains. Enterohaemorrhagic E. coli O157 strains were isolated from shellfish and sediment samples (7.07%). Multiplex PCR analysis revealed the presence of shiga toxin genes (stx1, stx2), intimin (eae) gene and enterohemolysin gene (hlyA). The overall prevalence of toxigenic genes among the strains was 78.1%; in shellfish (84%), sediment (78.1%) and water (69%). Shiga toxin gene stx2, showed the highest prevalence (54.3%), followed by eae (36.2%), stx1(20%), and hlyA (6.7%) genes. To the best of our knowledge, the present study is the first report on the isolation of E. coli O157 from shellfish harvesting areas along the Indian coast. Higher levels of faecal contamination along with the presence of pathogenic E. coli may pose potential health hazards to the consumers.