A series of novel guanidines (6a–j) was synthesized from corresponding thioureas using catalytic mercuric chloride in DMF. The synthesized compounds were characterized by spectroscopic techniques and appraised for Jack Bean Urease (JBU) inhibition. All compounds (6a–j) exhibited strong potential against JBU whilst compound 6b (IC50=0.0155 0.00087 μM) and 6e (IC50=0.0091 0.00036 μM) displayed excellent urease inhibition compared to thiourea (IC50= 18.27 μM) used as reference. The kinetic mechanism analyzed by Lineweaver-Burk plots revealed that 6b is a mixed-type inhibitor while 6e is a non-competitive inhibitor. Thus, compounds 6e and 6b can serve as a structural model for the designing of super-effective urease inhibitors. Binding affinity and interaction of N-aryl guanidine analogs were evaluated through molecular docking studies. To evaluate the residual flexibility of receptor through MD simulation, RMSD and RMSF graphs were evaluated to determine the protein structural behavior. It is Implied that compound 6e can serve as a novel molecular template to medicinal chemists for designing more potent urease inhibitors.