A supercritical CO2 pilot plant was employed to extract rosemary (Rosmarinus officinalis L.) leaves and to thoroughly study the effect of time on the yield, composition and antioxidant activity of the different fractions produced. Six extraction assays were carried out, varying the fractionation procedure using a two-stage depressurization system coupled to the extractor vessel. The concentration of the main antioxidant compound in rosemary, i.e. carnosic acid, and the DPPH test were employed to evaluate the antioxidant power of the different fractions obtained. The goal was to develop a new fractionation scheme comprising two different periods of time, in which exhaustive extraction of the essential oil from the plant matrix was achieved while recovery of the antioxidant compounds was maximized.