¹“G. d’Annunzio” University of Chieti-Pescara, 66100 Chieti, Italy DOI : 10.29228/jrp.388 Helicobacter pylori is a Gram-negative neutrophilic pathogen, which can cause chronic gastritis, peptic ulcers, and gastric cancer in humans. Current therapeutic multi-options regimens suffer from an emerging bacterial resistance rate and poor patience compliance. To improve the discovery of compounds targeting bacterial alternative enzymes or essential pathways such as carbonic anhydrases (CAs). Two isoforms belonging to the α- and the β- classes, namely HpαCA and HpβCA, were cloned, purified and characterized. They act in a complex interplay with urease. We have assessed the anti-H. pylori activity of some important plant secondary metabolites such as thymol, carvacrol, eugenol, and vanillin in terms of CA inhibition, isoform selectivity, growth impairment, biofilm production, and release of associated outer membrane vesicles-eDNA. In addition, chemical modifications were performed to improve the pharmacodynamic and pharmacokinetic profile. Cytotoxicity was also evaluated against human fibroblast (normal cell line) and AGS (cancer cell line). The microbiological results comprehended the evaluation in vitro of H. pylori CA inhibition, in silico analysis of the structural requirements to display such isoform selectivity, and the assessment of their limited toxicity against three probiotic species with respect to amoxicillin. Plant secondary metabolites could thus be considered as new lead compounds as alternative H. pylori CA inhibitors or to be used in association with current drugs for the management of H. pylori infection and limiting the spread of antibiotic resistance. Keywords : Carvacrol, thymol, eugenol, vanillin, Helicobacter pylori, carbonic anhydrase