This study aimed to evaluate the nutritional value and potential health claims for reduced sugar biscuits containing extruded brewers’ spent grain (EBSG) and fructooligosaccharides (FOS). One traditional biscuit with added sugar and three reduced sugar biscuits containing 15.2 % FOS and EBSG (0, 8 and 17 %), with nutrition claims “high in fiber” and “source of protein” for those containing 17 % of EBSG, were formulated. Biscuits were characterized by analysis of nutrients and bioactive compounds before and after digestion under in vitro enzymatic oral-gastro-intestinal and colonic fermenting conditions. The bioaccessibility of antioxidants, anti-inflammatory and antidiabetic compounds in biscuits’ intestinal digests and short-chain fatty acids in colonic samples was analyzed. EBSG-added biscuits showed significantly lower (p < 0.05) glucose intestinal bioaccessibility and significantly higher (p < 0.05) phenolic compounds intestinal bioaccessibility compared to biscuits without EBSG. EBSG-added biscuits showed significantly (p < 0.05) higher in vitro antidiabetic potential than the other did. Moreover, the intestinal digest of biscuits containing 17 % EBSG exhibited significantly (p < 0.05) better in vitro inhibition of intracellular ROS formation and in vitro anti-inflammatory properties. FOS addition (p < 0.05) significantly improved the production of butyric acid while EBSG did for valeric acid which possess chemoprotective effect. In conclusion, the combined use of FOS (15.2 %) and EBSG (17 %) allowed obtaining a human healthier snack formulation for satisfying consumers’ demands and achieving nutrition security.