Prunus fruit seeds are one of the main types of agri-food waste generated worldwide during the processing of fruits to produce jams, juices and preserves. To valorize this by-product, the aim of this work was the nutritional analysis of peach, apricot, plum and cherry seeds using the official AOAC methods, together with the extraction and characterization of the lipid profile of seed oils using GC-FID, as well as the measurement of the antioxidant activity and oxidative stability using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging method. Chemometric tools were required for data evaluation and the obtained results indicated that the main component of seeds were oils (30-38%, w). All seed oils were rich in oleic (C18:1n9c) and linoleic (C18:2n6c) acids and presented heart-healthy lipid indexes. Oil antioxidant activity was estimated in the range IC50 = 20-35 mg·mL-1, and high oxidative stability was observed for all evaluated oils during 1-22 storage days, with the plum seed oil being the most antioxidant and stable over time. Oxidative stability was also positively correlated with oleic acid content and negatively correlated with linoleic acid content. Therefore, this research showed that the four Prunus seed oils present interesting healthy characteristics for their use and potential application in the cosmetic and nutraceutical industries.