Brewers’ spent grain (BSG) is the insoluble residue generated from the production of wort in the brewing industry. This plant-derived by-product is known to contain significant amounts of valuable components, which remain unexploited in the brewing processes. Therefore, it is essential to develop a more detailed characterization of BSG in order to highlight its potential in developing new value-added products and simultaneously solve the environmental problems related to its discharge. The content of BSG in several biologically active compounds (fatty acids, polyphenols, flavonoids, antioxidant capacity) as well as its volatile fingerprint were assessed and compared with the composition of barley, malt and wheat flour samples. The obtained results emphasized the importance and the opportunities of the re-use of this agro-industrial by-product.