Lentinus giganteus is a edible mushroom cultivated in Asian countries. The present study was initiated to evaluate the anti-inflammatory, anti-acetylcholinesterase, anti-α-glucosidase, and free radical scavenging activities from fruiting bodies of L. giganteus extracted with methanol and hot water. The free radical scavenging activities of methanol and hot water extracts on 1,1-diphenyl-2-picrylhydrazyl (DPPH) were 92.26% and 90.17% at 2.0 mg/mL concentration, respectively and comparable with positive control, BHT. The chelating activities of methanol and hot water extracts were significantly higher than the positive control tested. The reducing power of methanol and hot water extracts showed lower activities compared to positive control, BHT. The phenolic and flavonoid contents of hot water extract were 1.56 μg/mg and 24.35 μg/mg, respectively. Nitric oxide (NO) produced by lipopolysaccahride (LPS) activated RAW 264.7 cells were significantly inhibited by treatment of methanol and hot water extracts. The methanol extract inhibited the acetylcholinesterase (AChE) activity by 91.19% at the 2.0 mg/mL concentration, whereas galanthamine, the standard drug, inhibited the AChE activity by 97.80%. The hot water extracts inhibited the α-amylase and α-glucosidase activities by 78.86% and 80.78%, respectively at the 2.0 mg/mL concentration, while acarbose, the positive control, inhibited the activities by 89.91% and 81.81%, respectively at the same concentration. Therefore, it is concluded that fruiting bodies of L. giganteus contain antioxidant, anti-inflammatory, anti-cholinesterase, and anti-diabetic substances, which can be used for natural health food for promoting human health.