Abstract: In order to analysis the chemical compounds and the biological activity of volatile oil from Lenzites betulina, the supercritical CO₂ and GC−MS were used to extract and analysis the volatile oil, and its antioxidant activity and antibacterial activity were also evaluated. The results showed that there are 31 compounds were identified from the volatile oil, and in which the content of linoleic acid was the highest(12.9%), and other main compounds were tetratriacontane(9.4%), tetracosane(6.4%), L−Ascorbic acid 2, 6−dihexadecanoate(4.5%), 9(11)−Dehydroergosteryl benzoate(3.2%), Icosane(2.3%), heneicosane(2.3%) and 2, 4−Di−tert−butylphenol(2.2%). The volatile oil from L. betulina showed strong antioxidant activity, and the antioxidant activity was linearly correlated with the concentration. The EC₅₀ values of the antioxidant activities against DPPH·, O₂⁻·, ·OH and ABTS⁺· were (115.81 ± 4.75), (40.72 ± 3.71), (185.26 ± 5.22) and (687.63 ± 6.53) μg/mL, respectively. The volatile oil also had the significant inhibition effects on Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris) and Gram-positive bacteria(Staphylococcus aureus subsp. aureus, Bacillus subtilis and Micrococcus tetragenus), which had the best bacteriostatic effect on Bacillus subtilis, and the minimum inhibitory concentration(MIC) was 3.91 μg/mL.