Antimicrobial resistance is considered a major threat facing humanity. It doesn't only affect public health, but also causes great losses in the dairy industry. Mastitis is a major threat to the dairy industry. The aim of this study was to monitor the antimicrobial resistance of Escherichia coli (E. coli) collected from raw milk of both healthy and mastitis cows and buffaloes in Egypt. In total, 450 milk samples were collected and examined in the period from 2018 to 2021. The samples were collected from healthy cows and buffaloes (30, 58), suffering from clinical mastitis (139, 223) respectively. E. coli was isolated from 33 mastitis milk samples (9.1%) and from 3 (3.4%) normal milk samples. The antibiotic susceptibility testing was performed using the disc diffusion method (Kirby-Bauer method). E. coli isolated from mastitis milk samples showed resistance to amoxicillin-clavulanic acid (75.8%), ceftazidime (75.8%), colistin (69.8%), ampicillin (39.4%), imipenem (24.2%), trimethoprim-sulfamethoxazole (24.2%), aztreonam (15.2%), cefotaxime (15.2%), tetracycline (15.2%), ciprofloxacin (12.2%), chloramphenicol (6%), and gentamicin (3%). E. coli isolated from normal milk samples showed resistance to ampicillin, and imipenem (66.7%), each, and amoxicillin-clavulanic acid, tetracycline, and trimethoprim-sulfamethoxazole (33.3%), each. The Extended Spectrum -Lactamases test (ESBL) performed on the E. coli isolates showed positive results in 9% of mastitis milk samples, but no results in normal milk samples. Out of 36 E. coli isolates, 34 possessed the ampC gene, but blaTEM and blaSHV were detected in 5 isolates with percentages of 94%, 1.4%, and 1.4%, respectively, while BlaIPM and Sul1 were found in one isolate (2.7%), for each.