To further elucidate antioxidant compounds of Eucalyptus leave extract, four compounds were isolated and purified from the fraction P95 of eucalyptus leave extract by medium-pressure silica gel column chromatography and preparative high performance liquid chromatography (HPLC). And three of the compounds were identified as hyperoside, guava glycosides and quercetin-3-o- alpha-pyranol-2"-gallate, by comparison of their 1H-NMR and 13C-NMR data. Then 1,1-diphenyl-2-picrylhydrazyl (DPPH·), 2,2‘-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS+·), and oxygen radical absorbance capacity (ORAC) assays were used for analyzing the antioxidant activity of the compounds. Among the DPPH and ORAC methods, guava glycosides showed the highest antioxidant activity, and quercetin-3-o- alpha-pyranol-2"-gallate showed the lowest antioxidant capacity. Hyperoside had the highest antioxidant activity, and guava glycosides had the lowest in ABTS+·assay. All of three compounds showed higher antioxidant activity than trolox, and they were natural antioxidants with better development prospects. This study provided a theoretical guidance for the comprehensive development of the Eucalyptus leaves.