Calf diarhoea is a main source of pre-weaning sickness in cattle operations. This study attempted to isolate Escherchia coli (E.coli) from calf diarhoea as well as evaluation of its role by analyzing the incidence of shiga toxin genes. Samples examined by culturing, VITEK test as well as molecular screening of E. coli virulence genes. The biochemical evaluation showed that the isolates were positive for Indole, Methyle red, Nitrate reduction, Lactose, and Arabinose sugar fermentation tests, while Voges Proskuaer, Citrate utilization, Urease, and H2S tests were negative and these results confirmed using VITEK® 2 compact as the isolate gave 98 % similar to the features of E. coli that recognized by the standard card for gram negative. From the examined samples 95 (63%) considered negative while only 55 (37%) were contaminated with E. coli. Only ten E. coli strains tested positive for O157, according to serological analysis. The genetic examination of stx (1&2) genes demonstrated that three samples identified E. coli O157 exhibited both stx (1&2) genes, one of them exhibited stx1, and two isolates carried stx2 only, whereas four E. coli O157 strains did not exhibit any stx genes. The total isolates of E. coli O157 obtained from calf diarrhea included rfbE and phoA genes.