Escherichia coli O157 strains cause diseases in humans that result from the consumption of food and water contaminated with faeces of infected animals and/or individuals. The objectives of this study were to isolate and characterise E. coli O157 strains from humans, cattle and pigs and to determine their antibiotic resistant profiles as well as detection of virulence genes by PCR. Eight hundred faecal samples were analysed for typical E. coli O157 and 76 isolates were positively identified as E. coli O157 strains. 16S rRNA sequence data were used to confirm the identity of the isolates. Susceptibility profiles to 9 antibiotics were determined and the multiple antibiotic resistant (MAR) patterns were compiled. A large proportion (52.6%-92.1%) of the isolates from pigs, cattle and humans were resistant to tetracycline, sulphamethoxazole and erythromycin. Thus the phenotype Smx-T-E (sulphamethozaxole-tetracycline-erythromycin) was present in most of the predominant MAR phenotypes obtained. Cluster analysis of antibiotic resistances revealed a closer relationship between isolates from pig and human faeces than cattle and humans. PCR were performed to amplify STEC virulence and tetracycline resistance gene fragments. A tetB gene fragment was amplified among the isolates. Eighteen (60%) of the isolates possessed the hlyA gene and 7(23.3%) the eae gene while only 5(16.7%) possessed both genes. Although shiga toxin genes were detected in the E. coli O157:H7 positive control strain none of the isolates that were screened possessed these genes. In a related study we reported that the prevalence of E. coli O157 was higher in pigs than cattle and humans. A high market demand for pork and beef in South Africa amplifies the risk that diseased animals pose to human health. This highlighted the need for proper hygiene management to reduce the prevalence of E. coli O157 in farm animals and prevent the spread from animals to humans.