Bovine subclinical mastitis was one of the most important health problems facing dairy industry, its impact exceeded the economic aspects and extended to potential negative effects on human health. The current study aimed to determine the prevalence of E. coli as an important mastitic pathogen and identify some of its most important virulence gene as well as their antimicrobial resistance profile. In the present study E. coli was isolated and biochemically identified whereas out of 100 subclinically mastitic milk samples was nine samples were positive for E. coli with 9% prevalence rate. Serotyping of these isolates declared that 3 isolates were serotype O26:H11, 2 isolates in serotype O91:H21 and 1 isolate in each of serotypes O55:H7, O128:H2, O146:H21 and O124. Antimicrobial resistance profile of the obtained isolates showing that all the isolates were 100% resistant to both erythromycin and streptomycin, while 88.9% (8/9) were sensitive to gentamicin. The presence of 3 important virulence factors including shiga toxin1(stx₁), shiga toxin 2 (stx₂) and intimin (eae) genes, among the obtained isolates was reported using PCR. Molecular investigation revealing that 2 isolates contain all studied virulence genes (stx₁, stx₂ and eae), 3 isolates contain (stx₁ and stx₂), while stx₁ was detected solely in 2 isolates, also 1 isolate contain only stx₂ and lastly 1 isolate was negative for any of the studied virulence factors. In a conclusion, there was a 9% prevalence rate of E. coli in subclinically mastitic milk samples in the current study, indicating its importance as a mastitic pathogen. The shiga toxin genes (stx₁ & stx₂) are widely distributed among E. coli isolates, while the intimin (eae) gene is less prevalent in comparison to shiga toxin genes. Also the recorded high multidrug resistance rate among the isolates posing threat to human health though entrance of these strains into the human being food chain whereas the isolated E. coli strains had the highest resistance to erythromycin and Streptomycin (100%), followed by Clindamycin (77.8%), Nalidixic acid (66.7%), and Gentamicin (11.1%) was the lowest.