Ethanol extract of the aerial portion of Chelidonium majus L. inhibited acetylcholinesterase (AChE) activity without a significant inhibition of butyrylcholinesterase (BuChE). Using mass spectrometry and NMR studies, three active constituents were isolated and identified: 8-hydroxydihydrochelerythrine (1), 8-hydroxydihydrosanguinarine (2), and berberine (3). Compounds 1—3 showed potent inhibitory activity against AChE, with IC₅₀ (μM) values of 0.61—1.85. Compound 1 exhibited competitive and selective inhibition for AChE.