Three hundred fifteen bacteriological samples were obtained from feces and both external and visceral cavity surfaces of carcasses of 105 healthy buffalo slaughtered in southwest of Iran. Confirmed Escherichia coli isolates were examined for antimicrobial resistance phenotypically and were screened for stx(1), stx(2), and eae genes and their subtypes and assessment of antimicrobial resistance genes by regular PCR and RFLP techniques. One hundred forty-five E. coli were isolated from feces (96 isolates) and external (37) and internal (12) surfaces of carcasses. Results showed that the prevalence of STEC, EPEC, and EHEC pathotypes was 2.8%, 0.7%, and 0.7% respectively. Among 6 (4.13%) positive isolates for examined genes, 4 (2.8%) isolates were positive for stx1, 3 (2.1%) for stx2, and 2 (1.4%) for eae gene. The detected genes were classified into stx(1)a (4 isolates), stx(2)a, stx(2)b, stx(2)c, eae-, and unknown subtypes. The most prevalent antibiotic resistance gene was sulII (11.03%). The tetB, qnrB, floR, bla(TEM), bla(SHV), and aad(A) genes were found to a lesser extent, and all isolates were negative for bla(CTX-15), bla(OXA), aac(3)-I, tetA, cat1, qnrA, sulI, dhfrI, and dhfrV genes. Twelve combination patterns of antibiotic-resistant genes were observed. Maximum phenotypically resistance rate was against doxycycline (91.83%), and the minimum was against ceftazidime and florfenicol (2.75%). E. coli isolates from feces and carcasses of slaughtered buffalo can be considered a mild reservoir for stx and eae genes. However, healthy buffaloes could be considered a potential reservoir of multiple antibiotic resistance genes in E. coli isolates.