Fish is a major vehicle for transmission several bacterial diseases to human. Escherichia coli (E. coli) is one of the main causes of food poisoning outbreaks occurring in Egypt due to consumption of contaminated fish and fish products. This study was performed to investigate prevalence of contamination of Oreochromis niloticus (O. niloticus) sold in the retail markets at Sohag Governorate, Egypt with E. coli and Shiga toxin-producing E. coli (STEC) and to determine the antimicrobial susceptibility of the isolates. Therefore, 110 apparently healthy O. niloticus samples were randomly collected from the fish retail markets at Sohag Governorate cities, Egypt and they were bacteriologically examined for presence of E. coli on the surface and in the gills and muscles. Susceptibility of E. coli isolates were tested to 10 different antibiotics and subsequently representative E. coli isolates (n=12) were serotyped and screened by PCR for presence of stx1 and stx2 genes. 22 E. coli isolates were isolated and identified from surface, gills and muscles of the examined O. niloticus with percentage of (8.2%), (10.0%) and (1.8%) respectively, these isolates were isolated from 12 fish only of the examined O. niloticus with total prevalence of 10.9% (12/110). Serotyping revealed that the investigated E. coli isolates belonging to 5 different O-serogroups comprising O55 (33.4%), O125 (25.0%), O26 (8.3%), O76 (8.3%) and O128 (8.3%) in addition to 2 nontypeable isolates (16.7%).