The aim of this study is to identify the role of E.coli in bovine mastitis, its virulence factors and antimicrobial sensitivity. 360 lactating cows and buffaloes were examined clinically and by CMT, 210 were suffering from clinical mastitis with percentage of 62.7% and 39 (20.36%) E.coli isolates were detected in clinical cases of mastitis, 90 cases were suffering from subclinical mastitis and 6 (6.8%) E.coli isolates were detected in them. The percentage of E.coli in cows was 31 (21.142%) and in buffaloes 8 (18.33%) isolates. Serotyping of E.coli revealed that O55 (30%), O111 (15%), O124 (15%), O119 (10%), O114 (10%), O26 (5%), O157 (10%) and O44 (5%). That’s showing that O55, O111 and O124 the most serotypes causing mastitis. PCR identification of TraT gene revealed 5 cases were positive and give positive reaction at 307 bp. and for eaeA gene revealed 6 cases and give positive reaction at 384 bp. The antimicrobial sensitivity indicated that the most effective antibiotics were lincospectine (56.6%), danofloxacin (56.6%), enrofloxacin (40%) and ceftifure (40%), while the lowest effective antibiotics were oxytetracycline and ampicillin.