Antioxidant activity of Australian tea tree (Melaleuca alternifolia) oil (TTO) was determined using two different assays. In the 2,2-diphenyl-1-picrylhydrazyl assay, 10 microL/mL crude TTO in methanol had approximately 80% free radical scavenging activity, and in the hexanal/hexanoic acid assay, 200 microL/mL crude TTO exhibited 60% inhibitory activity against the oxidation of hexanal to hexanoic acid over 30 days. These results were equivalent to the antioxidant activities of 30 mM butylated hydroxytoluene in both tests at the same experimental conditions. This indicated that the TTO could be a good alternative antioxidant. Inherent antioxidants, i.e., alpha-terpinene, alpha-terpinolene, and gamma-terpinene, in the crude TTO were separated and identified chromatographically using silica gel open chromatography, C(18)-high-pressure liquid chromatography, and gas chromatography-mass spectrometry. Their antioxidant activities decreased in the following order in both assays: alpha-terpinene > alpha-terpinolene > gamma-terpinene.