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Of the three types of waste generated in beer processing, brewer's spent grain (BSG) is the most abundant and has a high potential for valorization. In this work, defatted BSG (DB) was subjected to an extraction process with subcritical water at different temperatures to obtain extracts rich in phenols and the cellulosic fractions, which were also purified by using hydrogen peroxide (H2O2). The results showed that the dry extracts obtained at 170 °C were richer in phenolics (24 mg Gallic Acid Equivalent (GAE) g−1 DB), but with lower antioxidant capacity (71 mg DB·mg−1 2,2-diphenyl-1-pikryl-hydrazyl). This extract also showed the highest antibacterial potential against L. innocua (80 mg·mL−1) and E. coli (140 mg·mL−1) than those obtained at lower temperatures. The purification of cellulose from the treated residues, using hydrogen peroxide, revealed that DB is a limited source of cellulose material since the bleached fractions showed low yields (20–25%) and low cellulose purity (42–71%), even after four bleaching cycles (1 h) at pH 12 and 8% H2O2. Despite this, the subcritical water extraction method highlights the potential of a simple process as a technological option to convert underutilized side streams like beer bagasse into added-value, potential ingredients for innovative food and pharmaceutical applications. [ABSTRACT FROM AUTHOR]