Agave lechuguilla is a common plant of Northern Mexico that can be used as feedstock in the context of a biorefinery without competition for food use. In this work, the production of fermentable sugars from this biomass has been studied for the first time using dilute sulfuric acid pretreatment. An experimental design and response surface methodology were applied with temperature (160–200 °C) and acid concentration (0.5–1.5% w/v) chosen as factors. The pretreatment conditions were expressed in a combined severity factor, which ranged from −0.75 to 2.38. According to an optimization criterion that maximizes hemicellulosic sugar recovery in the prehydrolysate and glucose recovery by enzymatic hydrolysis, optimal conditions for acid pretreatment of agave were found to be 180 °C and 1.24% (w/v) H2SO4 at 10% biomass loading. These optimal conditions yielded 87% hemicellulosic sugar recovery and 68 g glucose/100 g glucose in raw agave. The whole slurry resulting from acid pretreatment of agave at optimal conditions was enzymatically saccharified yielding a sugar solution that was co-fermented by the ethanologenic Escherichia coli MM160. This process configuration allowed the fermentation of all sugars in raw A. lechuguilla in a single step reaching an ethanol yield of 73.3%.