Inonotus obliquus, an edible and medicinal mushroom parasitic on birches, has been used in human diet and for traditional therapies in the high latitude regions of Europe and Asia for a long time. Our phytochemical study of this fungus led to the identification of fourteen triterpenoids including four undescribed ones, and two pairs of undescribed phenolic enantiomers. The undescribed compounds were elucidated by extensive spectroscopic analysis including 1D and 2D NMR and HRESIMS, quantum chemical NMR and ECD calculations, as well as single-crystal X-ray diffraction analysis. Bioassays revealed that eight compounds showed dual inhibition against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) with IC50 values ranging from 2.40 ± 0.05 to 28.72 ± 0.46 μM, while 3β-hydroxy-lanosra-8,24-dien-21-al and trametenolic acid only presented BuChE inhibitory activities with IC50 values of 22.21 ± 1.01 and 7.68 ± 0.13 μM, respectively. In the kinetic studies, the most active three compounds acted as non-competitive inhibitors for both cholinesterases. Furthermore, molecular docking simulations revealed that three compounds demonstrated dual-sites bounding to AChE/BuChE. These triterpenoids emerged as bivalent and dual inhibitors of AChE/BuChE and could be effective drug candidates to prevent and treat Alzheimer's disease in the future. Phytochemical studies on Inonotus obliquus lead to identify bivalent and dual Inhibitors of acetylcholinesterase/butyrylcholinesterase. Display omitted •Fouteen triterpenoids including four undescribed were identified from Inonotus obliquus.•Two pairs of undescribed phenolic enantiomers were identified from Inonotus obliquus.•Three triterpenoids acted as noncompetitive inhibitors for AChE and BuChE.•Three triterpenoids demonstrated dual-site bound to AChE/BuChE.•One triterpenoid presented inhibitory effect on NO production in RAW264.7 cells.