Background: The increasing rate of water-borne diseases in Karachi demands the characterization of associated pathogens. Nowadays, water-borne infections become more resistant to treatment due to biofilm formation and excessive use of antibiotics. Biofilm formation in water deteriorates the quality of water. This study identified and characterized biofilm-producing E. coli in drinking water. Methodology: Water samples were analyzed for heterotrophic plate count and total coliforms, fecal coliforms, and E. coli. The Kirby disk bar diffusion method was used to investigate antibiotic susceptibility in biofilm-producing E. coli. The Congo red and tube ring methods were used to identify biofilm producers. The effect of biofilm formation on the hydrophobicity of E. coli was performed by the BATH method. The soft agar method determines the colony spreading ability of biofilm and non-biofilm producers. Molecular characterization of virulence genes of E. coli was performed by a PCR. Scanning electron microscopy for biofilm construction was conducted.   Results: The total 120 water samples were tested for heterotrophic plate count and total coliforms, fecal coliforms, and E. coli. 78% were unfit in this study, and 21.66% were fit. 38 E. coli strains were found in water samples. According to findings, the hydrophobicity of biofilm-producing isolates increased with the incubation period. Colony-forming unit drops one logarithm in the biofilm state compared to the planktonic stage. Biofilm producers were more resistant to antibiotics. The virulence genes, pet, lt, and stx2, were used for the molecular characterization. Conclusion: The presence of biofilm-producing E. coli in drinking water is alarming, and it indicates inappropriate treatment of the water supply system. To prevent rapid water-borne diseases, adequate actions are required to control drinking-water biofilm producers.