Luteolin (1), apigenin-7-0-13-beta-glucoside (2), luteolin 7-beta-13-D-glucoside (3), 6-OH-luteolin glucoside (4) were isolated from Achillea millefolium L. These flavonoids and different polarity fractions of methanol extract from aerial parts of the plant were investigated in terms of antioxidant and anticholinesterase effect. The n-butanol fraction possessed the highest inhibitory activity against acetylcholinesterase (51.3%) while lower inhibitory activity against butyrylcholinesterase (23.5%) as compared with a reference compound, neostigmine (100 and 98.5%), at 25 pg/mL. Compound 4 was exhibited in slum and in vitro the strongest inhibition to AChE and BuChE with IC50 values of 1.65 and 1.97 mu M, respectively and also have strongest antioxidant capacity. Docking simulations revealed that the most potent inhibitor compound 4 targeted both the catalytic and the peripheral active sites of AChE and BuChE with many hydrogen bond interactions. The ethyl acetate fraction has the richest phenolic content (224.6 GAE/mg ext). The mean amount of total flavonoids was 1.65% (calculated as rutin). (C) 2015 Elsevier B.V. All rights reserved.