Brewer’s spent yeast (BSY) is produced by the beer industry and has high nutritional value and great potential for producing high-value molecules, such as peptides, for nutraceutical, food and feed applications. In the present research, Flavourzyme® and Protamex® enzymes were selected for protein hydrolysis based on previous studies. The optimum conditions for the enzymatic hydrolysis were defined by response surface methodology (RSM) by the Box–Behnken design composed of four variables: temperature, pH, enzyme dosage and time. Protein content, hydrolysis degree and the anti-microbial and antioxidant bioactivities of obtained hydrolysates were quantified. Obtained results show that time, enzyme dosage and pH had the highest effect on protein extraction yield (PEY), degree of hydrolysis (DH) and antioxidant activity. Response variables ranged from 13.7 to 29.7% for PEY, from 6.3 to 35.7% for DH and from 0.65 to 1.65 g for Trolox equivalent antioxidant capacity. Antimicrobial activity, measured as minimum inhibitory concentration, against Aeromonas salmonicida, Bacillus cereus, Bacillus subtilis and Salmonella enterica, ranged from 6.25 to 50 mg/mL. Antioxidant and antimicrobial activity showed the potential use of BSY hydrolysates as an ingredient for functional foods.