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Mastitis is one of the most important health problems of dairy cattle as it causes physical, chemical and bacteriological changes in the produced milk, so the aim of this study is investigating the epidemiology of mastitis pathogens of dairy cattle and their associated risk factors. A total of 397 samples were gathered from mastitic cows and their surrounding environment including 127 milk samples from sub-clinically mastitic cows, 60 milk samples from clinically mastitic cows, 60 teat swabs, 50 milking machines swabs, 50 worker’s hand swabs and 50 bedding samples from different farms containing small and large dairy herds located in El Dakhalia and Menoufiya Governorate. These samples were examined bacteriologically and biochemically for isolation and biochemical identification of E. coli and S. aureus. Our results revealed that the prevalence of E.coli was 33.33% (11/33), 21.74% (20/92), 16.67% (4/24), 18.52% (5/27), 13.33% (2/15) and 13.33% (4/30) in milk samples from clinically mastitic cows, milk samples from subclinically mastitic cows, teat swabs, milking machines swabs, worker’s hand swabs and bedding samples, respectively, while the prevalence of Staphylococcus aureus was 23.08% (9/39), 17.43% (19/109), 11.9% (5/42), 10.53% (4/38), 17.07% (7/41) and 10.53% (4/38), in the same groups respectively. A significant relationship was found between of the number of these bacteria and cows age, herd size and hygienic condition of the farm. Moreover, PCR was done on 14 isolates of E. coli for detection of ESBL producing E. coli. The results of PCR revealed that the prevalence of blaTEM and blaSHV was 64.3% (9/14) and 92.9 …