Identification of compounds which can inhibit the activity of urease enzyme is an important approach towards the treatments of peptic ulcer, urolithiasis, and other urease associated disorders. This manuscript describes the in vitro urease inhibitory activity of different derivatives of phenols, heterocyclic compounds, thiols, quinone, tosylphenyl alanine, sulfonyl cyanides, phenyl acetoacetate, and thiamines (1-14). Among them, compound 10 was found to be more active with IC50 of 12.40 ± 1.57 µM, in comparison to the standard drug, acetohydroxamic acid (IC50= 41.5 ± 1.50 µM). Compounds 8 (IC50 = 21.0 ± 1.16 µM), and 9 (IC50= 24.94 ± 1.13 µM), were also identified as potent inhibitors of urease enzyme. All active compounds were largely found to be non-cytotoxic against fibroblast cell line (3T3 cell line). All potent compounds were also subjected to the mechanistic studies, as well as ligand-binding study by employing STD-NMR spectroscopy. In brief, this mechanism- based study identifies potent inhibitors of urease enzyme in vitro, as leads for further studies.