This study was planned to determine sources of contamination of E. coli O157 that are mainly discovered in animal, children's feces, milk, and water samples and serve as reservoirs for contamination by E. coli O157. About 500 samples (40 cattle fecal swabs, 150 sheep fecal swabs, 120 children fecal swabs, 100 milk samples, and 90 drinking water) were collected for bacteriological examination and identified by the Vitek2 system beside molecular detection of 16 isolates of E.coli O157 by PCR as well as detection of virulence genes (stx1, stx2, and eae) for STEC and EPEC isolates. Out of 500 examined samples 222 (44.4%) were positive for E. coli, by the Vitek2 system 16 (7.2%) isolates were positive for E. coli O157 (7.2%) with high incidence in cattle samples (20%), water samples (12.5%), children fecal samples (12.5% & 10%) but low percent (7.9%) in raw milk and (1%) in sheep. Molecular identification of 16 isolates of E. coli O157 by PCR detected 9 stx1, 6 stx2, and 16 eae-positive strains. It could be concluded that its important to put a control strategic plan for contamination by E. coli O157 on farms and hospitals to minimize the incidence of different infections in animals and humans.