A series of organic extracts obtained from spines, flowers, roots and fruits of two Tunisian species of Opuntia, O. microdasys var. rufida (OMR) and O. leptocaulis (OL) were investigated for their phytochemical composition as well as their neuroprotective, anticholinesterase, cytotoxic and catalase activities. The anticholinesterase activity was assessed using butyrylcholinesterase (BChE) as the plasma cholinesterase which revealed that all extracts were endowed of an excellent inhibitory efficacy against BChE. The neuroprotective effect of the prepared extracts was evaluated against ?-amyloid-induced toxicity on PC12 cell lines in vitro, the methanolic extract of OMR’s spines increased significantly cell viability against ?-amyloid-induced cytotoxicity and reached 80%. The MTT assay shows that a few extracts displayed obvious cytotoxic activity against HeLa cells, the strongest inhibition percentage was 62.42%. Furthermore, the antioxidant activity of the obtained extracts was evaluated by the catalase test, the results demonstrate that all the extracts were very potent and the values range from (112.32 µmol of H2O2 destroyed/min/mg protein) to (749.12 µmol of H2O2 destroyed/min/mg protein). However, the EtOAc extract of OMR’s flowers displaying the highest anti-BChE effect (IC50 = 0.04 ± 0,00 mg/ mL) was subjected to a column chromatography to afford five flavonol glycosides identified as isorhamnetin-3-O-?-rhamnopyranosyl-(1 ? 2)[?-rhamnopyranosyl-(1 ? 6)]-? galactopyranoside (1), quercetin-3-O-?-pyranogalactoside (hyperoside 2), isorhamnetin-3-O-?-galacto (1 ? 6)-?-rhamnoside (3), isorhamnetin-3-O-?-glucoside (4) and kaempferol-3-O-?-arabinoside (5). The docking of the isolated 1-5 compounds in the BuChE’s active site revealed their possible inhibitory effect on the catalytic activity of the enzyme. It has been found that the most favorable docking results were obtained with compound 3.< Réduire