The aim of this study was to investigate the in vitro antioxidant and antimicrobial activities of acetone, ethanol, and ethyl acetate extracts from Flammulina velutipes. Antioxidant activity was evaluated based on total contents of polyphenols and flavonoids, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, and 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity. Total contents of polyphenols and flavonoids were the highest in ethyl acetate extracts. The DPPH radical scavenging activities of these extracts were in the order of ethanol (82.3%)> ethyl acetate (79.6%)> acetone (78.4%)> ascorbic acid (76.8%). Ethanol extract showed significantly different (P<0.05) DPPH radical scavenging activities compared with other extracts. ABTS radical scavenging effects were 74.7% and 73.0% for acetone and ethanol extracts, respectively. Antimicrobial activities of F. velutipes extracts were evaluated by the paper disk diffusion method against six strains of microbes, including Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa, and Enterobacter cloacae. Acetone and ethyl acetate extracts showed antimicrobial effects against all tested strains, and especially ethyl acetate extract had strong antimicrobial activities against M. luteus and E. coli. Antioxidant activities were negatively correlated with contents of total polyphenols (-0.192 with DPPH and -0.953 with ABTS) and total flavonoids (-0.717 with DPPH and -0.704 with ABTS). A positive relationship was established among total flavonoid contents and DPPH and ABTS radical scavenging activities. This study confirms positive antioxidant and antimicrobial activities for F. velutipes. © 2018, Korean Society of Food Science and Nutrition. All rights reserved.