Urease catalyzes the hydrolysis of urea to carbamate and ammonia, leading to nitrogen loss, environmental pollution, and health issues, so numerous compounds have been screened for urease inhibition using Jack bean urease (JBU) and H. pylori urease (HPU) without consideration their structure difference. Previous studies have shown that the same inhibitor can exhibit distinct inhibitory effects on JBU and HPU, but limited papers focus on the effects mechanism. In this study, we systematically investigated the thermodynamic and kinetic properties of JBU and HPU binding with quercetin, focusing on the structural effects on both commonly studied ureases. The results revealed that quercetin inhibited both JBU and HPU activities, with IC