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In this study, supercritical fluid extraction has been successfully applied to a sequential fractionation of fatty acids and polyphenols from wine wastes (2 different vitis vinifera grapes). To this aim, in a 1st step just fatty acids were extracted and in a 2nd one the polyphenols. The variables that affected to the extraction efficiency were separately optimized in both steps following an experimental design approach. The effect of extraction temperature flow, pressure, and time were thoroughly evaluated for the extraction of fatty acids, whereas the addition of methanol was also considered in the case of the polyphenols extraction. A quantitative extraction with high efficiency was achieved at a very short time and low temperatures. Concerning quantification, fatty acids were determined by means of gas chromatography coupled to mass spectrometry after a derivatization step, whereas the polyphenols were analyzed by means of high performance liquid chromatography coupled to tandem mass spectrometry and the Folin–Ciocalteu method.