A microwave assisted hydrothermal pretreatment technology has been developed to recover fermentable sugars from brewer’s spent grain. Microwave hydrothermal pretreatment is considered as a greener pretreatment, as no acid or alkali are used as catalysts. An experimental design was planned to analyze the effect of pretreatment conditions (temperature and time). The objective was to maximize hemicellulosic sugar recovery in the liquid fraction and glucose recovery in enzymatic hydrolysis (referred to untreated BSG), as well as to minimize inhibitors in the liquid fraction to ensure ABE (acetone, butanol and ethanol) fermentability. Optimal conditions were 192.7 °C and 5.4 min, resulting in 64% hemicellulosic sugar recovery, 70% glucose recovery in enzymatic hydrolysate and 2.4 g/L total inhibitors. The liquid fraction obtained under optimal conditions was fermented with Clostridium beijerinckii, reaching a butanol concentration as low as 1 g/L. The butanol concentration could be improved by operating at higher solid loadings in pretreatment, which would increase fermentable sugar concentration. Enzymatic hydrolysis of pretreated BSG yielded a sugar solution, which was also fermented, resulting in a butanol concentration and overall yield of 8.3 g/L and 46 kg/t BSG, respectively.