Water is one of the crucial necessities of man since it is needed for almost all of his activities. With increase in population growth, quality water needed for consumption by man is on the decline because of environmental pollution. Thus the main aim of this study was to investigate the prevalence level of E. coli O157:H7 in water bodies in Ile-Ife and its environs which are the sources of water for man’s consumption. Four hundred and fifty-one samples were investigated from five major sources of water namely; well, stream, borehole, bottled and sachet. The water samples were screened for E. coli and serologically characterized for E. coli O157:H7. The E. coli O157:H7 isolates obtained were further characterized for stx 1, stx 2 and eaeA genes. The result revealed that well water and stream water had a prevalence of 8.74% and 4.59% respectively. Antibiotic resistance profile of E. coli isolates showed that both sources harboured isolates that are resistant to three classes of antibiotics. The virulence gene distribution shows that stx 1 and stx 2 were present in all the E. coli O157:H7 investigated. In conclusion, it appears that the gastroenteritis outbreaks experienced in the study area may be due to the presence of E. coli O157:H7 in water samples.