Second-generation bioethanol is considered a suitable option for replacing fossil fuels. Agricultural residues are being studied as feedstocks for sugar generation, which are in turn converted into ethanol. Among them, barley straw (BS) is a promising raw material, due to its high abundance, lignocellulosic composition and lack of other practical applications. Under these assumptions, the central aim of this study is to suggest an efficient bioethanol production scheme from BS at different levels of integration in co-fermentation with Escherichia coli SL100, including separate hydrolysis and co-fermentation (SHCF), simultaneous saccharification and co-fermentation (SSCF), and presaccharification and simultaneous saccharification and co-fermentation (PSSCF), using the water-insoluble solid (WIS) and slurry fractions obtained after steam explosion (SE) pretreatment. The best results in terms of ethanol yield were achieved following the SHCF process, using the WIS and the slurry as substrates, with yields of 89.1% and 78.8% of the theoretical maximum, respectively. Considering all of the above points, the following scheme is proposed for the conversion of BS into ethanol: SE pretreatment (160 °C, 30 min) of BS previously soaked overnight in 2.88% w/v phosphoric acid solution, filtration of the slurry, followed by enzymatic hydrolysis and co-fermentation of the two fractions obtained separately, with previous detoxification of the prehydrolysate with ammonium hydroxide (5 N). Under these conditions, 19.43 g of bioethanol was produced from 100 g of BS.