Taiwo Olayemi Elufioye,1 Efere M Obuotor,2 Joseph M Agbedahunsi,3 Saburi A Adesanya4 1Department of Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Ibadan, 2Department of Biochemistry, 3Drug Research and Production Unit, Faculty of Pharmacy, 4Department of Pharmacognosy, Faculty of Pharmacy, Obafemi Awolowo University, Ile Ife, Osun, Nigeria Abstract: Spondias mombin has been used in traditional medicine for the management of several diseases, including memory loss. This study aimed to evaluate the cholinesterase inhibitory activity of the methanol extract of the leaves and its derived fractions, as well as carry out detailed phytochemical investigations leading to the isolation and characterization of bioactive compounds from the plant. The acetyl cholinesterase (AChE) and butyryl cholinesterase (BUChE) inhibitory activities were evaluated by colorimetric and thin-layer chromatography bioautographic assay techniques. The ethyl acetate fraction was most active against both enzymes, with percentage inhibition of 58.10 ± 1.08% and 52.66 ± 1.34% against AChE and BUChE, respectively. Three compounds, namely, botulin, campesterol and phytol, with IC50 of 0.88 μg/mL (AChE), 4.67 μg/mL (BuChE); 1.89 μg/mL (AChE), 4.08 μg/mL (BuChE) and 12.51 μg/mL (AChE), 23.89 μg/mL (BuChE), respectively, were isolated from the supernatant of the ethyl acetate fraction. The isolated cholinesterase inhibitory compounds correlate with the known memory-enhancing property of the plant and thus support one of its uses in ethnomedicine. Keywords: Alzheimer’s disease, acetylcholinesterase, butyryl cholinesterase, neurodegenerative, Spondias mombin