In this work, the co-production of xylosaccharides and butanol from Eucalyptus grandis wood using steam explosion pretreatment in a biorefinery platform was investigated. The effect of different temperature conditions on xylosaccharide production and selectivity as well as enzymatic hydrolysis of pretreated E. grandis substrates was studied using a pre-pilot steam explosion reactor. Under selected conditions (200 ◦C, 1.5 MPa, 10 min), steam explosion pretreatment led to about 50% xylan recovery as xylooligosaccharides (XOS) and xylose and 80% glucan enzymatic hydrolysis even at high solid loading. A xylosaccharide-rich hydrolysate was effectively separated by ion-exchange and resin treatment, containing mostly xylose and xylobiose as XOS. The fermentation performance of four native butanol-producing strains (Clostridium acetobutylicum DSM 1731, Clostridium beijerinckii DSM 6422, Clostridium beijerinckii DSM 6423 and Clostridium saccaroperbutylacetonicum DSM 14923) were evaluated using semi-synthetic media and enzymatic hydrolysates for butanol production. Results showed that ABE strain Clostridium beijerinckii DSM 6422 and IBE strain Clostridium beijerinckii DSM 6423 are robust native strains for butanol production from lignocellulosic materials. This work highlights a potential integrated biorefinery process for the efficient utilization of E. grandis wood to produce biofuels and biochemicals.