Background and Aim:Vietnam’s dairy sector is in its early phase of large-scale farming development. Therefore, mastitis in cows is always a concern to farm owners. This study aimed to determine the antimicrobial susceptibility, resistance, and virulence-related genes ofEscherichia coliisolated from bovine mastitis in Nghe An province of Vietnam.Materials and Methods:FiftyE. colistrains were isolated from the clinical cases and subjected to this study. All isolates were tested for antimicrobial susceptibility by the disk-diffusion method, as described by the Clinical and Laboratory Standards Institute. Antimicrobial and virulence genes were confirmed by polymerase chain reaction with specific primers.Results:All isolates were resistant to lincomycin and sulfamethoxazole and sensitive to gentamicin, while other antimicrobials showed resistance from 2% to 90%. Multidrug resistance was confirmed in 46% of isolates, and none of them were identified as extended-spectrum beta-lactamase producers. From fifty strains tested for antimicrobial and virulence genes, six isolates harboredtetA, 6tetB, 13sul1, 15sul2, 2 Intimin (eae), 1iutA, and 3stx2.Conclusion:Antimicrobial and multidrug resistances are the main virulence factors ofE. coliisolated from bovine mastitis in Vietnam. The virulence genes encoding adhesion, siderophore, Shiga-toxin-producing, and antimicrobials resistant were first reported in Vietnam with low prevalence and contributed to the pathogenesis.