The increase in the prevalence of antibiotic resistance in Helicobacter pylori had a drastic effect on successful treatment. Up-to-date information on H. pylori antibiotic therapy in Iran is still limited. In this study, we aim to determine the prevalence of antibiotic resistance among the H. pylori strains. Furthermore, the possibility of using fluoroquinolones for antibiotic treatment was investigated. Antral biopsy specimens obtained from dyspeptic patients were investigated for H. pylori. Bacterial culture and susceptibility tests were done based on standard methods. H. pylori ATCC 43504 was used as a quality control. In the current study, 30 H. pylori strains were selected randomly and retested to confirm our susceptibility tests. Of 170 patients, 150 were identified as positive for H. pylori (88.2%). In this study, 150 single colonies of H. pylori strains [81 women (54%), 69 men (46%); mean age 38.6; aged 21-70 years] were collected. Primary resistance of H. pylori isolates were clarithromycin (34%), metronidazole (78.6%), tetracycline (9.3%), amoxicillin (10%), levofloxacin (5.3%) and moxifloxacine (4.6%). In conclusion, our results show that we are confronting a new generation of resistant strains of H. pylori in Iran. This alarming finding indicates an urgent need for introduction of new effective antibiotics in our country. Since the majority of clinicians prefer to continue with the ineffective antibiotics as therapeutic regimens, they must also be prepared to deal with treatment failures.