The present study aimed to evaluate the antioxidant activity of petroleum ether, methanolic extracts and active ingredients separated from Eucalyptus globulus using three different antioxidant assays: 2,2 diphenyl picryl hydrazyl (DPPH), 2,2’- azino-bis [ethylbenzthiazoline-6-sulfonic acid] (ABTS) and β-carotene bleaching assay and identify the mode of action. The results revealed that, crude methanolic extract showed higher antioxidant activity against both DPPH and ABTS radicals than petroleum ether extract. The promising methanol soluble fraction of Eucalyptus globulus wood was fractionated on a silica gel column, using hexane, chloroform and ethyl acetate as the mobile phase to give three fractions (C1, C2 and C3), and both the antioxidant activity and chemical composition for raw and fractions were determined. One of the fractions isolated (C2) showed a remarkable antioxidant activity (EC50 of 64.4
µg/ml, in comparison with 52.74 µg/ml for crude extracts) against ABTS radical method, and the chemical structures of separated active ingredients were identified using different spectroscopic methods such as 17-pentatricontene (C1), N,N-diphenyllauramide (C2) and O-benzyl-N-tert-butoxycarbonyl-D-serine (C3). Also, the mode of action of the promising fraction was determined.