Although Escherichia coli (E. coli)is a harmless gut microbe in man, some strains of this bacterium are pathogenic due to the acquisition of virulence factors. The aim of this study isto investigate E. coli O157:H7 strains isolated from Ogun River, Abeokuta metropolis, for virulence factors and antibiotic resistance. Water samples were collected bimonthly from six different locations over a period of six months. The samples were cultured on Sorbitol MacConkey Agar and E. coli O157:H7 isolates were confirmed through serological characterization using the latex agglutination test. The presence of virulence genes (stx1, stx2, eae, and hylA) in the isolates was analyzed through polymerase chain reaction (PCR). Further, antibiotic susceptibility of the isolates was tested using the disc diffusion method. The PCR analysis revealed that the five E. coli O157:H7 strains isolated possessed Shiga toxin 1 (stx1), Shiga toxin 2 (stx2), and Haemolysin (hlyA) genes. Additionally, the isolates were resistant to Augmentin, Ceftriaxone, Nitrofurantoin, Gentamycin, Amoxicillin, and Pefloxacin. This study shows that E. coli O157:H7 strains are present in Ogun River and that these strains possess multiple virulence factors and are resistant to multiple drugs.