F main phenolic compounds were isolated from the mushroom Inonotus obliquus by a repeated column chromatography. The structures of the five phenolic compounds were identified as 3,4-dihydroxybenzaldehyde (1),vanillic acid (2), caffeic acid (3), syringic acid (4) and 3,4-dihydroxybenzalacetone (5) using 1H and 13C NMR in combination with mass spectrometry. A simple reversed-phase high performance liquid chromatography (RP-HPLC) procedure was developed for simultaneous determination of the five bioactive phenolic compounds. Three extraction methods on Inonotus obliquus extract were compared and reflux extraction was the efficient method with the strong antioxidant properties. There are stronger linear relationships between DPPH radical scavenging activity and total phenolic content, caffeic acid and 3,4-dihydroxybenzalacetone content (R2>0.95). Caffeic acid and 3,4-dihydroxybenzalacetone are the major contributors to the observed antioxidant activities. This could be important information for quality control and the structure-activity relationships of phytochemicals of mushroom Inonotus obliquus.