This study falls within the framework of the industrial exploitation of by-products of the prickly pear (Opuntia ficus-indica). The study aims to evaluate the use of hydro-ethanolic extract of prickly pear peels as a substitute of vitamin E used as antioxidant in margarine preservation. The extract was rich in total phenolics (1512.58 mg GAE/100 g DM). HPLC-DAD-ESI-MSn analyses allowed the identification of sixteen compounds belonging to hydroxybenzoic acids, hydroxycinnamic acids and flavonoids. The extract displayed a reducing power and an antiradical activity that were respectively similar to and lower than the two antioxidant standards quercetin and butylated hydroxyanisole. Tests conducted at laboratory and pilot scales showed that the margarines elaborated with peel extract were more resistant to oxidation than the margarine reference with vitamin E. In addition, neither the physicochemical nor the microbiological properties were modified. Prickly pear peels contain bioactive substances that could be used in different food sectors.