Background: Early detection of Helicobacter pylori (H. pylori) infection is essential for its treatment. Resistance to amoxicillin, clarithromycin and metronidazole has been on the increase in many countries. Phenotypic resistance is correlated with treatment failure. So, there is an urgent need to explore sensitivity of other antibiotics, such as levofloxacin to combat H. pylori infection. Objective: The study was aimed to detect H. pylori from gastric biopsy samples and its susceptibility profile to commonly used antimicrobial drugs. Methods: Gastroduodenal biopsy specimens were collected from 143 adult dyspeptic patients during March 2018-February, 2019, who attended the outpatient department of gastroenterology, Bangabandhu Sheikh Mujib Medical University (BSMMU) and Dhaka Medical College Hospital (DMCH), for endoscopy. H. pylori was identified by rapid urease test (RUT), ureC gene PCR, histological staining (Giemsa) and culture. From culture isolates antimicrobial susceptibility of clarithromycin, levofloxacin, amoxicillin and metronidazole were detected by disk diffusion method. Results: The highest rate of H. pylori infection was found in the age group between 41-50 years (25.5%). According to case definition, H. pylori positive cases were 47 (32.9%) and H. pylori negative cases were 96 (67.1%). Thirty five H. pylori positive samples were subjected to culture and only 10 (28.6%) were positive. Among 10 culture positive H. pylori isolates, clarithromycin exhibited 20% resistance, levofloxacin 30%, metronidazole 30% and no resistance found to amoxicillin. Conclusion: PCR based assays can be an alternative rapid approach for the detection of H. pylori. In this study, levofloxacin showed high resistance, a further larger study is required to confirm this finding. Bangladesh Med Res Counc Bull 2022; 48(1): 3-9