A new series of arylmethylene hydrazine derivatives bearing 1,3-dimethylbarbituric moiety 7a-o were designed, synthesized, and evaluated for their in vitro urease inhibitory activity. All the title compounds displayed high anti-urease activity, with IC50 values in the range of 0.61 ± 0.06-4.56 ± 0.18 µM as compared to the two standard inhibitors hydroxyurea (IC50 = 100 ± 0.15 μM) and thiourea (IC50 = 23 ± 1.7 μM). Among the synthesized compounds, compound 7h with 2-nitro benzylidene group was found to be the most potent compound. Kinetic study of this compound revealed that it is a mix-mode inhibitor against urease. Evaluation of the interaction modes of the synthesized compounds in urease active site by molecular modeling revealed that that compounds with higher urease inhibitor activity (7h, 7m, 7c, 7l, 7i, and 7o, with IC50 of 0.61, 0.86, 1.2, 1.34, 1.33, 1.94 μM, respectively) could interact with higher number of residues, specially Arg609, Cys592 (as part of urease active site flap) and showed higher computed free energy, while compounds with lower urease activity (7f, 7n, 7g, and 7a with IC50 of 3.56, 4.56, 3.62 and 4.43 μM, respectively) and could not provide the proper interaction with Arg609, and Cys592 as the key interacting residues along with lower free binding energy. MD investigation revealed compound 7h interacted with Arg609 and Cys592 which are of the key residues at the root part of mobile flap covering the active site. Interacting with the mentioned residue for a significant amount of time, affects the flexibility of the mobile flap covering the active site and causes inhibition of the ureolytic activity. Furthermore, in silico physico-chemical study of compounds 7a-o predicted that all these compounds are drug-likeness with considerable orally availability.