This study investigated the antioxidant potential and anti-fatigue effects of phenolics extracted from the seed coat of Euryale ferox Salisb. The in vitro antioxidant potentials, including scavenging DPPH, hydroxyl radical activities and reducing power were evaluated. Antioxidant status in vivo was analyzed by SOD, CAT, GSH-Px activities and the MDA content in liver and kidneys of D-galactose-induced aging mice. The anti-fatigue effect was evaluated using an exhaustive swimming test, along with the determination of LDH, BUN and HG content. The phenolic extract possessed notable antioxidant effects on DPPH, hydroxyl radical scavenging and reducing power. The mice which received the phenolic extract showed significant increases of SOD, CAT (except for in the kidney), GSH-Px activities, and a decrease of MDA content. The average exhaustive swimming time was obviously prolonged. Meanwhile, increase of LDH content and decrease of BUN content were observed after mice had been swimming for 15 min. The HG storage of mice was improved in the high and middle dose extract groups compared with the normal group. The contents of total phenols and gallic acid of the extract were determined. Three compounds in the extract were identified as 5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-chroman-4-one, 5,7,4-trihydroxyflavanone and buddlenol E. These results suggest that the extract of E. ferox is a promising source of natural antioxidants and anti-fatigue material for use in functional foods and medicines.