Bovine mastitis is a common worldwide infection caused by different types of pathogens including coliform bacteria. Understanding mastitis pathogens and their dominance, as well as risk factors, are required to facilitate disease prevention and control and to enhance udder health within the herd. Our objectives were to investigate the prevalence of different mastitis-causing pathogens and describe some genetic traits which outline mastitis-associated E.coli (MAEC). Four-hundred twenty-eight quarter foremilk samples were collected from 107 apparently healthy lactating cows and buffalo from El-Menofia governorate between 2020-2022. The California Mastitis Test (CMT) was used to estimate the quarter somatic cell count (SCC), with subclinical mastitis (SCM) defined as non-negative CMT score. The bacterial culture of milk was used as a reference method to identify SCM based on the isolation of the causative pathogens. VITEK-2 compact system was used for isolates identification. Serological identification of E.coli serotypes and molecular identification of some virulence genes using PCR was also performed. The prevalence of SCM was 60.7% with E.coli being the most commonly isolated organism. The enteropathogenic serotypes of E.coli isolated from the examined milk samples showed; O26:H11 (EHEC) 21.7%, O15:H2 (ETEC) 13.04%, O127:H6 (ETEC) 4.34%, O121:H7 (EHEC) 13.04%, O117:H4 (EHEC) 17.39%, O146:H21 (EPEC) 17.39% and O103:H2 (EHEC) 13.04%. The molecular identification of the most common virulent gene of E.coli revealed the presence of sfa, papC and traT genes in all examined samples while cnf1 gene is present in only 30% of the samples.