In the current study, the virulence factors in Escherichia coli isolates from bovine mastitis were investigated, and the connection between these factors and infection was evaluated using phenotypic and genotypic analyses. Twenty-seven E. coli isolates were analyzed, and 2 were shown to produce verotoxin. All isolates had the ability to produce biofilms, although at different levels. One isolate was found to be sensitive to the bactericidal activity of bovine serum, 11 were intermediate, and 15 were resistant. Some isolates showed resistance to trimethoprim sulfa (9) and ampicillin (4), intermediate resistance to neomycin (1) and trimethoprim sulfa (5), and simultaneous resistance to ampicillin and trimethoprim sulfa (4). The fimH gene was found in all isolates and was associated with other virulence markers: pap (1), stb (8), cs31a (3), stb and vt2 (2), cs31a and stb (3), east1 and kps (1), stb and east1 (1), cs31a and east1 (1), and cs31a, stb, pap, and iucD (1). Serogroups were determined for 3 isolates: O93:H4, O83:H19, and O15:H11. Phylogenetic analysis showed that 23 isolates belonged to group A and 4 belonged to B1. The findings revealed that these E. coli isolates are opportunistic pathogens with different virulence factors. The results indicate that the pathogenicity route of E. coli in bovine mastitis is not a consequence of 1 specific virulence factor.