Impaired cholinergic system is an important therapeutic target to develop drugs against Alzheimer's disease (AD), as cholinergic signal transduction is associated with the memory, learning, and behavior of an individual. Acetylcholine (ACh) is an important neurotransmitter and is degraded by an enzyme, acetylcholinesterase (AChE), blocking neuronal signal transmission, which leads to amnesia or memory loss. Oxidative stress (OS) also worsens AD pathology by damaging lipids, proteins and DNA of the neuronal cells, leading to neuronal cell death. Golden oyster mushroom (GOM), an edible mushroom native to the regions of Japan, Korea, Russia, and China, has been reported to possess several bioactivities. However, there is no study reported till date, studying the anti-amnesic properties of GOM. The objectives of this study were to assimilate the in vitro neuroprotective and in vivo anti-amnesic properties of GOM. The GOM at 0.1, 0.5, 1, 5, and 10 mg/ml was nontoxic and was able to prevent HO (500 µM) and glutamate (5 mM)-induced OS-mediated cell death in HT-22 cells. In addition, all the selected concentrations of GOM extract (50, 100, and 200 mg/kg, 28 days) were able to prevent scopolamine (ACh receptor antagonist, 1 mg/kg, 1 h post-treatment after the 7, 14, and 28 administrations)-induced cholinergic system impairment, memory loss, histopathological alteration, antioxidant defense system disruption, and reduced memory susceptibility markers in C57BL mice compared to the positive control tacrine (10 mg/kg, 28 days). In conclusion, GOM extract emerged as a natural AChE inhibitor and could be seen as a potential therapeutic AD agent.