Among the waste materials of wine production, grape seeds constitute an important fraction of the pomace, from which the precious edible oil is extracted. The residual mass from oil extraction, the defatted grape seeds (DGS), can be destined for composting or valorized according to the circular economy rules to produce pyrolytic biochar by gasification or pellets for integral energy recovery. Only a small quantity is used for subsequent extraction of polyphenols and tannins. In this study, we performed a chemical characterization of the DGS, by applying spectroscopic techniques (ICP-OES) to determine the metal content, separation techniques (HS-SPME-GC-MS) to evaluate the volatile fraction, and thermal methods of analysis (TGA-MS-EGA) to identify different matrix constituents. Our main goal is to obtain information about the composition of DGS and identify some bioactive compounds constituting the matrix in view of possible future applications. The results suggest that DGS can be further exploited as a dietary supplement, or as an enriching ingredient in foods, for example, in baked goods. Defatted grape seed flour can be used for both human and animal consumption, as it is a source of functional macro- and micronutrients that help in maintaining optimal health and well-being conditions.