Abstract In this work, a dilute sulfuric acid pretreatment at mild conditions was employed to fractionate brewer’s spent grain (BSG) with the aim of achieving high carbohydrate recovery. A Box-Behnken experimental design was used with temperature (110–130 °C), acid concentration (1–3% w/v) and time (10–40 min) as independent factors, the objective being to determine the best conditions for the release of glucose (following enzymatic hydrolysis of the pretreated solids) along with that of hemicellulosic sugars in the liquors. The optimal pretreatment conditions were found to be 130 °C, 1% H2SO4 and 26 min, which allowed the recovery of 94% of the sugars in raw BSG. Next, the effect of substrate concentration on the simultaneous saccharification and fermentation (SSF) process of the pretreated solid was studied. Likewise, the fermentability of the resulting prehydrolysate was evaluated with two xylose fermenting microorganisms, Scheffersomyces stipitis and Escherichia coli. The overall proposed BSG bioconversion process yielded 22.9 L bioethanol from 100 kg of dry biomass.