Machilus thunbergii was an important medicinal resource and distributed widely in China. In this study, the bioactivities of dichloromethane fraction (DF) and water fraction (AF) from Machilus thunbergii methanol extract were investigated. Total phenolic contents of DF and AF were 57.90 mg Gal/g and 189.92 mg Gal/g, and total flavonoid contents were 17.34 mg Que/g and 58.38 mg Que/g respectively. The EC50 for DPPH radical scavenging activity of DF and AF were 24.37 μg/ml and 2.10 μg/ml. Reducing power and hydroxyl radical scavenging activities of AF were higher than those of DF. The α-glucosidase inhibitory activity of AF (IC50 = 1.13 μg/ml) was higher than that of DF (IC50 = 5.34 μg/ml). The cell viability was showed that only the DF had anti-proliferation effect on human cancer cell HT-29. These results suggested that both the DF and AF extract of Machilus thunbergii were potential materials for anti-diabetes and functional food for their radical scavenging activity.