The current study was conducted to determine the role of E. coli in causing subclinical mastitis in sheep and to determine the role of bedding and water as sources of E. coli associated with environmental mastitis in sheep and goats. In addition, determination of the antibiotic sensitivity of some E. coli isolates to select the suitable antibiotics for treatment of mastitis and finally, application of PCR for detection of some virulence genes. Escherichia coli were isolated from milk, bedding and water of sheep farms at the rate of 8.67, 16 and 10%, respectively while it were isolated from milk, bedding and water of goats farms at the rate of 15.33, 24 and 10%, respectively. Moreover, Twenty E. coli isolates were serologically identified revealing the presence of serotypes; O114:H21, O111:H4 and O124. Results of antibiogram test performed for E. coli O114:H21 clarified that it was highly sensitive for Ciprofloxacin (CIP 5 µg), Enrofloxacin (5 µg) and Oxytetracycline (30 µg). E. coli isolates were tested by multiplex PCR for detection of stx1 (555 bp) and eaeA (425 bp) genes for characterization of Enteropathogenic E. coli successfully. Finally, it was concluded that, E. coli could be isolated from the examined samples of milk, bedding and water of different farms under investigation. Also, it was noticed that the isolation rates of bacteria was higher in goats farms compared to sheep farms which may be due to the defects in the hygienic conditions inside these farms.