Hydrothermal pre-treatments have been developed to decrease lignocellulosic biomass recalcitrance by solubilizing and disrupting the majority of the hemicellulose on the biomass thus increasing cellulase accessibility. However, a small quantity of the hemicellulose may still remain and become adsorbed to the cellulose, leading to cellulase inhibition. In this study, we produced hemicellulose bound cellulose, using glucuronoxylan and galactomannan, to simulate hydrothermally pre-treated hardwoods and softwoods, respectively, and evaluated how these may affect cellulose hydrolysis by Trichoderma reesei cellobiohydrolase I, Cel7A. Based on XRD, FTIR, SEM and Simon’s staining, hemicellulose binding onto cellulose affected the physical properties of the biomass, and consequently its hydrolysis rate. As a result of hemicellulose binding onto cellulose, the adsorption of Cel7A was significantly affected (up to a 42 and 60% reduction for xylan and mannan, respectively), leading to lowered activities (~40% reduction), especially for xylan. The bound hemicellulose may be released from the cellulose during agitation and hydrolysis; we therefore evaluated the effect of free hemicellulose on Cel7A activity. Xylan was more inhibitory to Cel7A than mannan, demonstrating non-competitive inhibition, while mannan demonstrated uncompetitive inhibition. The recalcitrance effect of bound hemicellulose could thus be completely relieved by the addition of hemicellulolytic enzymes (i.e. a Bacillus stearothermophilus T6 xylanase, XT6, and an Aspergillus niger mannanase, Man26A) during hemicellulose bound cellulose hydrolysis. This study showed that hemicellulose remains a critical factor regarding biomass recalcitrance and that the addition of hemicellulolytic activities in commercial enzyme cocktails is required, especially the mannanolytic activities lacking from most enzyme cocktails.