Urease inhibition potential of compound (1), guaiane-type sesquiterpene (2), confertin (3) and scopoletin (4) was carried out with high throughout mechanism-based assay. These compounds were isolated from Hypochaeris radicata L., an Asteraceae family member. The pure compounds were screened for their urease and carbonic anhydrase inhibitory activities. The ethyl acetate fractions were subjected to column chromatography, which resulted in the isolation and purification of four compounds (1-4). On evaluation, compounds (1-4) exhibited selective activity against urease enzyme with an IC 50 value of 180.11 ± 2.00, 27.18 ± 0.80, 24.12 ± 0.2 and 30.12 ± 1.10 µM respectively. The compounds (1-4) were found to be inactive against carbonic anhydrase enzyme. Thiourea was used as standard inhibitor (21 ± 0.14 µM) of urease enzyme.