Abstract: Urease is an amidohydrolase enzyme that is responsible for fatal morbidities in the human body, such as catheter encrustation, encephalopathy, peptic ulcers, hepatic coma, kidney stone formation, and many others. In recent years, scientists have devoted considerable efforts to the quest for efficient urease inhibitors. In the pharmaceutical chemistry, the thiourea skeleton plays a vital role. Thus, the present work focused on the development and discovery of novel urease inhibitors and reported the synthesis of a set of 1-aroyl-3-[3-chloro-2-methylphenyl] thiourea hybrids with aliphatic and aromatic side chains 4a–j. The compounds were characterized by different analytical techniques including FT-IR, 1H-NMR, and 13C-NMR, and were evaluated for in-vitro enzyme inhibitory activity against jack bean urease (JBU), where they were found to be potent anti-urease inhibitors and the inhibitory activity IC50 was found in the range of 0.0019 ± 0.0011 to 0.0532 ± 0.9951 μM as compared to the standard thiourea (IC50 = 4.7455 ± 0.0545 μM). Other studies included density functional theory (DFT), antioxidant radical scavenging assay, physicochemical properties (ADMET properties), molecular docking and molecular dynamics simulations. All compounds were found to be more active than the standard, with compound 4i exhibiting the greatest JBU enzyme inhibition (IC50 value of 0.0019 ± 0.0011 μM). The kinetics of enzyme inhibition revealed that compound 4i exhibited non-competitive inhibition with a Ki value of 0.0003 μM. The correlation between DFT experiments with a modest HOMO-LUMO energy gap and biological data was optimal. These recently identified urease enzyme inhibitors may serve as a starting point for future research and development.