Introduction Tomentosin, the characteristic component of Inula viscosa (L.) is an important sesquiterpene lactone with anticarcinogenic effects. Methods of obtaining pure tomentosin are not sufficient for anticancer drug research. Objectives This study aims to develop a specific method to isolate tomentosin from I. viscosa with high yield. It also aims to investigate the inhibitory effects of tomentosin on human carbonic anhydrase I (hCAI), human carbonic anhydrase II (hCAII), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α‐glucosidase, and α‐amylase enzymes. Material and methods Tomentosin was purified by a specific column chromatography method. The content of tomentosin in dichloromethane, dichloromethane by Soxhlet method, ethanol and ethanol by Soxhlet method extracts of I. viscosa was determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Half maximal inhibitory concentration (IC50) and inhibition constant (Ki) values were calculated to determine in vitro enzyme inhibition effects. Results Tomentosin was isolated in high yield (0.64%). The IC50 and Ki values for tomentosin were calculated as 5.00 ± 0.19 (r = 0.9688) and 4.62 ± 0.10 μM for hCAI, 5.40 ± 0.26 (r = 0.9677) and 5.22 ± 0.31 μM for hCAII, 6.75 ± 0.208 (r = 0.9891) and 3.75 ± 0.27 μM for AChE, 6.67 ± 0.307 (r = 0.9820) and 0.51 ± 0.11 μM for BChE, 26.61 ± 0.236 (r = 0.9815) and 2.61 ± 0.71 μM for α‐glucosidase and 26.89 ± 1.54 μM (r = 0.9670) for α‐amylase, respectively. Conclusion Tomentosin was isolated in high yield from the paste‐like extract of I. viscosa compared to the positive controls, it was determined that tomentosin was weakly effective against hCAI, hCAII, AChE and BChE, but thoroughly effective against α‐glucosidase and α‐amylase. These results suggested that tomentosin has α‐glucosidase and α‐amylase inhibitor potential.