The possibility of using oregano leaves as a new source of natural antioxidants has been investigated. In the present study, oregano leaves were extracted using a pilot-scale SFE plant under a wide range of extracting conditions (at different extraction pressures and temperatures and considering the addition of ethanol as modifier) with the objective of knowing not only the best extraction conditions but also the variables that control the process in terms of extraction and fractionation of products of high antioxidant activity. The different fractions obtained at each experimental condition tested were characterized in terms of chemical composition by using liquid chromatography-diode array detection (LC-DAD) and liquid chromatography-mass spectrometry (LC-MS). Nine compounds have been tentatively identified as belonging to a specific family of compounds (flavone, flavanone and flavonol); their aglycon structures have been proposed using UV spectra profile while a tentative pattern of substitution, based on their mass spectra and molecular ion (MH+) major fragments, has been also given. Antioxidant activity was measured using two different in vitro assays (1,1-diphenyl-2-picrylhydrazyl (DPPH) antiradical test and bleaching beta-carotene method). (C) 2006 Elsevier B.V. All rights reserved.