The aims of this study were to investigate the presence of Escherichia coli O157 and to determine the antibiotic susceptibility of isolates in different poultry-originated materials. In total 300 samples of chicken carcass swabs (n = 50), chicken feces (n = 50), gizzard (n = 50), liver (n = 50), water in the final scald tank (n = 50), and equipment such as tables, conveyers, and knives (n = 50) were used as materials. Overall, 45 E. coli isolates were obtained from the 300 poultry samples. However, 13 (4.3%) of the samples were identified as E. coli O157 including 3 liver, 3 carcass, and 7 fecal samples. All 13 isolates were positive for O157 but negative for H7 using PCR. Only intimin (encoded by eaeA gene) was detected in 10 E. coli O157 isolates while no other virulence factors (enterohemolysin A, shiga toxin 1 and 2) were detected. All E. coli O157 isolates were susceptible to gentamicin (100%). In conclusion, the presence of E. coli O157 in poultry is of utmost significance (4.3%) and our results demonstrated that poultry carcasses and giblets may be contaminated by E. coli O157 with feces during evisceration. To prevent E. coli O157 infections, hygiene rules must be tightly observed. In addition, antibiotic use must be controlled for avoiding increased antibiotic resistance.