Background:Helicobacter pylori (H. pylori) is one of the most common infectious diseases in the world. It colonizes about 50-60% of the world's population. The estimated prevalence of H. pylori infection is approximately 65% in Iran. Objectives: The aim of the present study was to investigate the antimicrobial resistance of H. pylori to metronidazole, clarithromycin and amoxicillin. Materials and Methods: A total of 110 biopsy specimens were collected from patients with clinical symptoms of gastrointestinal disorders who were referred to the endoscopy unit at Al-Zahra Hospital, Isfahan, Iran. We investigated the frequency of H. pylori resistance to metronidazole, clarithromycin and amoxicillin among 48 positive isolates of H. pylori from the referred patients. After the culture of biopsy specimens and identification of H. pylori strains, susceptibility tests were done using the E test (epsilometer test) Results: The overall rates of resistance to metronidazole, clarithromycin and amoxicillin were 56.3%, 14.6% and 4.2% respectively. In our study, there was a significant correlation between resistance to metronidazole and gender (P < 0.05) but there was no significant correlation between resistance to amoxicillin and clarithromycin and gender (P ˃ 0.05). Conclusions: Information on antibiotic susceptibility profile plays an important role in empiric antibiotic treatment and management of refractive cases. With regards to this study, using metronidazole in the Isfahan region, can lead to eradication failure in clinical therapies due to having the highest rate of resistance but amoxicillin and clarithromycin are prescribed for first and second lines of treatment against H. pylori. Actually, susceptibility testing of H. pylori isolates in different geographical areas is advised because it aids the selection of optimal therapy regimens.