Background: Nearly 50 % of the world residents are known to be infected with Helicobacter pylori which is the main cause of peptic ulcer and gastric cancer. Since resistance to the currently used treatments, other alternative approaches such as combination with natural products should be tried. Objectives: In this work, validated HPLC-UV method was developed for quantification of lepidine content in various extracts and crude oil of Lepidium sativum seeds. Furthermore, antibacterial activity of lepidine against H. pylori was compared with standard antibiotics supported by molecular docking study. Methods: Different solvents were used to extract lepidine as well as the contents were determined using HPLC-UV method. Anti-Helicobacter pylori activity was examined using agar diffusion and dilution methods. Molecular docking study was done on H. pylori phosphoribosyltransferase enzyme. Results: The highest content of lepidine (316.91 µg mL^-1) was found in n-hexane extract. Lepidine exhibit antibacterialeffect (MIC = 6.25 µg mL^-1) against H. pylori clinical isolates. The ability of lepidine to interact with amino acids in the phosphoribosyl transferase binding site might rationalize its observed activity. Conclusion: Our results demonstrate for the first time the anti-Helicobacter pylori activity of lepidine, which could therefore be developed as viable nutraceutical agent. Further investigations are required to formulate suitable pharmaceuticals to combat with the H. pylori infections on clinical grounds.