AbstractCholinesterase (ChE) and monoamine oxidase (MAO) inhibitors have been attracted as candidate treatments for Alzheimer's disease (AD). Fifteen khellactone-type coumarins from the roots ofPeucedanum japonicumThunberg were tested for acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and MAO inhibitory activities. Compound 3′-angeloyl-4′-(2-methylbutyryl)khellactone (PJ13) most potently inhibited AChE (IC50 = 9.28 µM), followed by 3′-isovaleryl-4′-(2-methylbutyroyl)khellactone (PJ15) (IC50 = 10.0 μM). Compound senecioyl-4′-angeloyl-khellactone (PJ5) most potently inhibited BChE (IC50 = 7.22 μM) and had the highest selectivity index (> 5.54), followed by 3′-senecioyl-4′-(2-methylbutyryl)khellactone (PJ10) and 3′,4′-disenecioylkhellactone (PJ4) (IC50 = 10.2 and 10.7 μM, respectively). CompoundsPJ13,PJ15, andPJ5showed reversible and mixed-types of inhibition with Kivalues of 5.98, 10.4 (for AChE), and 4.16 µM (for BChE), respectively. However, all 15 compounds weakly inhibited MAO-A and MAO-B. Molecular docking simulation revealed thatPJ13had a higher binding affinity (− 9.3 kcal/mol) with AChE thanPJ15(− 7.8 kcal/mol) orPJ5(− 5.4 kcal/mol), due to the formation of a hydrogen bond with Tyr121 (distance: 2.52 Å). On the other hand, the binding affinity ofPJ5(− 10.0 kcal/mol) with BChE was higher than forPJ13(− 7.7 kcal/mol) orPJ15(− 8.1 kcal/mol), due to the formation of a hydrogen bond with Ser198 (distance: 2.05 Å). These results suggest thatPJ13andPJ5are potential reversible selective inhibitors of AChE and BChE, respectively, for the treatment of AD.