The carbonic anhydrase inhibitors acetazolamide (AAZ) and methazolamide (MZA) are safe, clinically used drugs originally designed to treat non-bacteria related illnesses (e.g. glaucoma). But they also show antimicrobial activity against the gastric pathogen Helicobacter pylori. AAZ in particular is sufficiently effective to promote ulcer healing and to prevent ulcer recurrence, indicative of its potential as an anti-H. pylori drug. In this study, the inhibitory activities of AAZ, MZA and ethoxzolamide (EZA) were measured against several H. pylori laboratory and clinical strains. Mutants resistant to these compounds were isolated, characterized, and their genomes sequenced. The results show that AAZ, EZA and MZA kill H. pylori via mechanisms that are different from the mode of action of common antibiotics used to treat H. pylori infections, as they retain activity against antibiotic-resistant clinical isolates. Acquisition of resistance to sulfonamides in the laboratory was associated with a complex phenotype and genetic mutations in up to 12 genes, including the one encoding undecaprenol pyrophosphate synthase, a known target of sulfonamides. The data suggest that sulfonamides impact multiple targets in killing H. pylori. The frequency of single-step spontaneous resistance acquisition by H. pylori was low, showing that resistance does not develop easily. The antimicrobial activity of these compounds is restricted to H. pylori and a few other bacteria, including Streptococcus pneumoniae, Neisseria spp. and Brucella suis. Our findings suggest that this class of approved drugs can be developed into selective anti-H. pylori agents with a novel mechanism of action.