Introduction: Resistance to many of the antibiotics used to treat Helicobacter pylori (HP) infection is on the rise. Indeed, the WHO has included H.pylori on their priority list of antibiotic-resistant bacteria to guide research and development into novel antimicrobials. To this end, protein-ligand docking of 550k+ compounds was carried out against the purine nucleoside phosphorylase enzyme (PNP), a key survival enzyme of HP. Aims: (i) perform in silico docking to identify compounds with potential activity against HP via PNP enzyme inhibition, (ii) and to test the in vitro antimicrobial and cytotoxicity activity of the compounds. Methods: The binding site of the PNP enzyme was computational analysed and a library of compounds was virtually screened via protein ligand docking to identify several lead-hits to carry forward to in vitro screening. Lead-hits were tested for antimicrobial efficacy against reference strains (J99 and ATCC60190) and clinical isolates of HP using a broth microdilution approach. Selectivity was established using a viability assay with a stomach epithelial cell line AGS. Results: Lead-hits were selected from protein-ligand docking results and tested in vitro. All compounds showed antimicrobial activity against the reference strains and both clarithromycin-sensitive and clarithromycin-resistant clinical isolates of HP (MIC 4.34-73 pg/mL). 2 compounds showed significant selectivity against human cells, having no activity on the viability of human gastric cells. Conclusion: Protein-ligand docking provided a cost-efficient method to identify selective antimicrobial agents for H. pylori resulting in the identification of several lead targets that may be further developed to increase selectivity and potency.