Regarding the important role of the urease enzyme as a virulence factor in urease-positive microorganisms in this study, new series of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives were designed and synthesized. All compounds evaluated against urease enzyme exhibiting IC50values of 0.87 ± 0.09 to 8.32 ± 1.21 µM as compared with thiourea as the positive control (IC50= 22.54 ± 2.34 µM). The kinetic evaluations of6aas the most potent derivative recorded a competitive type of inhibition. Molecular dynamic simulations of the6aderivative were also conducted, showing that6aoccupied the active site with closed state. Antimicrobial activities of all derivatives were performed, and6f(R = 3-Cl),6g(R = 4-Cl), and6h(R = 3,4-diCl) analogs demonstrated significant antifungal activities with MIC values of 1, 2, and 0.5 µg/mL compared with fluconazole with MIC = 2 µg/mL. Synthesized analogs also exhibited potent urease inhibitory activities againstC. neoformans(IC50= 83.7–118.7 µg/mL) andP. mirabilis(IC50= 74.5–113.7 µg/mL), confirming their urease inhibitory potential. The results demonstrated that the designed scaffold could be considered a suitable pharmacophore to develop potent urease inhibitors.