Degenerative diseases are commonly associated with excess free radicals. Acer okamotoanum, a plant endemic to Korea, is reported to have anti-oxidant, anti-cancer, and anti-viral activities. We previously isolated flavonoids from the ethyl acetate fraction of A. okamotoanum such as quercitrin (QU), isoquercitrin (IQ), and afzelin (AF). In the present study, the in vitro antioxidant activity of flavonoids such as QU, IQ, and AF isolated from the ethyl acetate fraction of A. okamotoanum were investigated by measuring the free radical scavenging activity including 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (•OH), and superoxide anion (O-). The flavonoids (QU, IQ, and AF) concentration-dependently showed a DPPH radical scavenging activity. In particular, QU and IQ showed a higher DPPH radical scavenging activity than that of AF. In addition, the flavonoids (QU, IQ, and AF) at 10 μg/mL showed over an 80% scavenging effect against •OH radical production. Furthermore, the O- radical scavenging activity of the flavonoids, QU, IQ, and AF increased in a dose-dependent manner. Particularly, IQ exerted the strongest scavenging activities against •OH and O- radicals among the other flavonoids. These results indicate that the flavonoids from A. okamotoanum, in particular IQ, would have a protective activity against oxidative stress induced by free radicals, and potentially be considered as a natural antioxidant agent.