Essential oil of Biebersteinia multifida DC cultivated in Iran was obtained by hydrodistillation (HD) and supercritical fluid extraction (SFE) methods. The oil was analyzed by capillary gas chromatography using flame ionization and mass spectrometric detections. The effects of different parameters such as temperature, pressure, static extraction time, dynamic extraction time, and mole fraction of modifier on the supercritical fluid extraction (SFE) and radical scavenging activity of B. multifida oil were optimized using central composite design. Fifty two compounds were identified in the HD oil. The major components of B. multifida were (E)-Nerolidol (31.45%), Hexadecanoic acid (11.84%), Phytol (17.1%), and 6,10,14-trimethyl-2-pentadecanone (15.4%). However, by using supercritical carbon dioxide in optimum conditions, obtained only four components contain more than 91.74% of the essential oil. The optimum extraction yield and DPPH EC50 via SFE method were 96% (w/w) and 27.19, respectively. (C) 2014 Elsevier B.V. All rights reserved.