Cow mastitis caused by Escherichia coli (E. coli) exhibits various local and systemic clinical signs at varying degrees of severity. The aim this study was to elucidate the virulence properties, antibiotic resistance and phylogenetics of 56 E. coli strains. Of all the studied strains, 12 were positive for hemolytic properties and 38 were positive for biofilm production. Additionally, 55 of the strains were positive for multiple resistances in bacteriological tests. PCR analysis revealed that 42 strains carried the traT gene, 20 strains had the shiga toxin gene (stx1-stx2), and 8 strains carried the intimin gene (eae), but all strains were negative for aerobactin gene (aer). All strains encoding shiga toxin genes were also positive for stx1, but only 4 strains were positive for stx2. There were no significant differences in virulence genes between antibiotic-resistant and antibiotic-susceptible strains. The random amplified polymorphic DNA-polymerase chain reaction patterns revealed the existence of 13 main groups with 4 subgroups of E. coli. In this study, E. coli strains causing intramammary infections and originating from various sources might show resistance against common antibiotics. Pathogenity of E. coli that cause clinical mastitis, and prognosis of the infection might be predicted by obtaining the traT gene. Additionally, antibiotic resistance should be investigated at the genomic level to detect the relationship between virulence factors and antibiotic resistance. In field conditions, development of antibiotic resistance is the main cause of mastitis treatment failure. Thus, antibiotic resistance profiles in herds should be monitored and effective antibiotics should be administered.