The involvement of high acetylcholinesterase (AChE) activity and oxidative stress in the brain is well documented in the progression of dementia. Thus, finding a drug with both AChE-inhibitory and antioxidant activities could be beneficial in treating dementia. We previously reported the AChE-inhibitory and antioxidant-mediated antiamnestic effects of a hydromethanol extract from Ganoderma mediosinense (HME), which we evaluated using in vitro and in vivo models. Mycochemical screening of HME showed the presence of phenols. Building on those findings, this study was designed to isolate the compound responsible for the AChE-inhibitory and antioxidant activities of G. mediosinense. The HME was fractionated sequentially with solvents-namely, hexane, chloroform, and ethyl acetate. The prepared fractions were evaluated by using Ellman and DPPH-inhibitory assays in vitro. Among the fractions, the ethyl acetate fraction showed appreciable AChE-inhibitory (half-maximal inhibitory concentration [IC50] 0.81 ± 0.04 mg/mL) and DPPH scavenging (IC50 2.04 ± 0.77 μg/mL) activities; therefore it was subjected to flash chromatography, which separated 9 subfractions. Subfraction 7 showed marked activity in inhibiting AChE (IC50 0.10 ± 0.02 mg/mL) and free radicals (IC50 1.22 ± 0.04 μg/mL). Purification of subfraction 7 yielded a white compound, AK1. This was characterized as gallic acid by using Fourier-transform infrared, nuclear magnetic resonance, and mass spectral studies. This study shows that gallic acid, a well-recognized phenolic acid, is responsible for the AChE-inhibitory and DPPH scavenging activities of G. mediosinense.