Urease enzyme is a virulence factor that helps in colonization and maintenance of highly pathogenic bacteria in human. Hence, the inhibition of urease enzymes is well-established to be a promising approach for preventing deleterious effects of ureolytic bacterial infections. In this work, novel thiobarbiturate derivatives were synthesized and evaluated for their urease inhibitory activity. All tested compounds effectively inhibited the activity of urease enzyme. Compounds 1, 2a, 2b, 4 and 9 displayed remarkable anti-urease activity (IC<sub>50</sub> = 8.21-16.95 μM) superior to that of thiourea reference standard (IC<sub>50</sub> = 20.04 μM). Moreover, compounds 3a, 3g, 5 and 8 were equipotent to thiourea. Among the tested compounds, morpholine derivative 4 (IC<sub>50</sub> = 8.21 µM) was the most potent one, showing 2.5 folds the activity of thiourea. In addition, the antibacterial activity of the synthesized compounds was estimated against both standard strains and clinical isolates of urease producing bacteria. Compound 4 explored the highest potency exceeding that of cephalexin reference drug. Moreover, biodistribution study using radiolabeling approach revealed a remarked uptake of <sup>99m</sup>Tc-compound 4 into infection induced in mice. Furthermore, a molecular docking analysis revealed proper orientation of title compounds into the urease active site rationalizing their potent anti-urease activity.