Raw milk plays an important role in the survival and transport of pathogenic bacteria including Escherichia coli strains. This study was performed to determine the existence of E. coli in raw milk intended for human consumption and its associated virulence determinants. In this context, a total of 232 milk samples were obtained from different dairy shops located at Mansoura city and its surrounding villages. Milk samples were subjected for bacteriological and serological examination of E. coli. Furthermore, E. coli strains were tested for its haemolytic activity on blood agar plates. The recovered E. coli strains were also screened by Polymerase chain reaction (PCR) for the presence of enterotoxins including heat –labile (LT), heat- stable (ST) toxins and haemolysin (hly) genes. The recovery rate of E. coli was 14.65% (34/232). Among the recovered E. coli strains, 12 different E. coli serotypes were identified namely, O26:H11 (n꞊6), O111:H2 (n꞊5), O128:H2 (n꞊5), O91:H21 (n꞊4), O124 (n꞊3), O127:H6 (n꞊3), O103:H21 (n꞊2), O153 (n꞊1), O113:H4 (n꞊2), O6 (n꞊1), O121:H7 (n꞊1) and O146 (n=1). Regarding PCR results, 31(91.19%) E. coli strains harbored STa and seven strains carried hly gene (20.59%) while non E. coli isoates harbord LT gene. Conclusively, raw milk can be considered as serious source of pathogenic E. coli, therefore, proper management practices and effective control measures are recommended to improve milk hygiene and sanitation.