Urease enzyme is a known therapeutic drug target for treatment ofHelicobacter pyloriinfection due to its role in settlement and growth in gastric mucosa. In this study, we designed a new series of sulfonates and sulfamates bearing imidazo[2,1-b]thiazole scaffold that exhibit a potent inhibitory activity of urease enzyme. The most potent compound2cinhibited urease with an IC50value of 2.94 ± 0.05 μM, which is 8-fold more potent than the thiourea positive control (IC50= 22.3 ± 0.031 μM). Enzyme kinetics study showed that compound2cis a competitive inhibitor of urease. Molecular modeling studies of the most potent inhibitors in the urease active site suggested multiple binding interactions with different amino acid residues. Phenotypic screening of the developed compounds againstH.pyloridelivered molecules of that possess high potency (1a, 1d, 1h, 2d, and2f) in comparison to the positive control, acetohydroxamic acid. Additional studies to investigate the selectivity of these compounds against AGS gastric cell line andE.coliwere performed. Permeability of the most promising derivatives (1a, 1d, 1h, 2d, and2f)in Caco-2 cell line, was investigated. As a result, compound1dpresented itself as a lead drug candidate since it exhibited a promising inhibition against urease with an IC50of 3.09 ± 0.07 μM, MIC value againstH.pyloriof 0.031 ± 0.011 mM, and SI against AGS of 6.05. Interestingly, compound1ddid not show activity against urease-negativeE.coliand exhibited a low permeability in Caco-2 cells which supports the potential use of this compound for GIT infection without systemic effect.