Coliform pathogens, primarily E. coli, were discovered throughout the farm, causing environmental mastitis and can be shed from the udder into the milk; they are concerned about severe gastrointestinal disruption and potential enteropathogenic and/or toxic strains, posing a risk to public health. The objectives of this study were to identify the incidence and harmful serotypes of pathogenic Escherichiacoliand some of their virulent genes, which were isolated from the collected milk of some dairy farms in the Delta region and Cairo-Alexandria desert road farms, in Egypt during one year using the polymerase chain reaction (PCR) technique after bacteriological and serological identification as well as determine the antimicrobial susceptibility of the isolated strains. 150 milk samples in total were gathered (100 milk samples from bulk milk tanks and the other 50 samples from clinically mastitic dairy cows). According to our finding, the mean values of somatic cell count (SCC), standard plate count (SPC), and coliform count (CC) in the hundred bulk tank milk samples were 3.67 1.08×104/ml, 7.08×104± 6.25×104 cfu/ml and 3.04×102±1.43×102/ml, respectively. The bacteriological investigation exhibited that, the Escherichiacoli incidences from bulk tank milk (BTM) and mastitic milk samples (MMS) were 12% and 18%, respectively. The detected E.coli serotypes including, O26, O44, O55:K99, O111, O119 and O157:H7 from MMS, while O1, O55, O78, O86, O124 and O158:H10 from BTM. Molecular virulence characterization of E.coli strains showed that, Shiga toxins 2 (stx2) gene is present in O157:H7, while the stx1 gene present in O26. The Intimin gene (eaeA) is involved in four strains, O44, O111, O119, and O157:H7. Positive amplification of a biofilm gene (adrA) appeared in all E.coli strains. The outcome of the antimicrobial susceptibility revealed high resistance to amoxicillin (85.71%), streptomycin (80.95%), ampicillin (71.43%), and flucloxacillin (61.90%). Meanwhile, the highest susceptibility was to ciprofloxacin (95.24%) followed by enrofloxacin (90.48%), neomycin (80.95%), and gentamycin (76.19%). Effective hygienic measurements are required to avoid toxigenic and pathogenic E.coli and more future studies should be performed to increase awareness in dairy farms.