Suillus luteus (L.) Roussel is an edible mushroom commonly known as slippery jack or “Kallampa” by indigenous people from Loja province. It is used in traditional medicine to manage gastrointestinal disorders and headaches. In addition, edible mushrooms have been used for neurodegenerative diseases; however, there is no report about the anticholinesterase effect produced by this species. The aim of this work was to isolate the main secondary metabolite of Suillus luteus and characterize its inhibitory potential against acetylcholinesterase. Fruiting bodies were extracted with ethanol (EtOH) and ethyl acetate (EtOAc). From the EtOAc, suillin, is reported as the major compound. The cholinesterase inhibitory potential of extracts and the major isolated compound was assessed by Ellman´s method and progression curves were recorded at 405 nm for 60 min. Donepezil hydroclhoride was used as a positive control. The samples were dissolved in methanol at 10 mg/mL and two more 10× dilutions were included to obtain final concentrations of 1, 0.1 and 0.01 mg/mL at the mix of reaction. IC50, Km, Vmax, and Ki were calculated for suillin. Suillin (200 mg) along with linoleic acid, ergosterol peroxide and ergosterol were isolated. The EtOH and EtOAc extracts exerted a moderate inhibitory effect (IC50 > 200 μg/mL. In adittion, suillin exerted a non-competitive mixed mechanism. against AChE with an IC50 value of 31.50 μM and Ki of 17.25 μM. To the best of our knowledge, this is the first report of the anticholinesterase effect of Suillus luteus and suillin. The kinetic parameters and the moderate potency of the compound determined in this study, encourage us to propose suillin as a promising chemopreventing agent for the treatment of neurodegenerative diseases such as Alzheimer.