Replacement of fossil fuels with renewable sources has been studied as a strategy to mitigate the environmental impacts of energy production. These concerns have encouraged the use of alternative sources of cost-competitive and sustainable biofuels, such as lignocellulosic biomass, for second-generation ethanol production. As the enzymes required for biomass hydrolysis are costly, the development of less expensive enzyme mixtures for the hydrolysis of sugarcane biomass is important. The efficient conversion of biomass to fermentable sugars requires a critical pretreatment step to increase enzyme accessibility to the substrates during hydrolysis. Ionic liquids (ILs) have recently emerged as interesting solvents for biomass pretreatment. Extensive research has been conducted, showing that IL pretreatment can increase fermentable sugar productivity and biomass hydrolysis yields in comparison with conventional pretreatments, such as dilute sulfuric acid (DSA). The objective of this work was to evaluate changes in the chemical composition of a sugarcane biomass mixture composed of straw and bagasse in a 1:1 (w/w) ratio after pretreatment with the IL 1-ethyl-3-methylimidazolium acetate ([Emim] [Ac]) or with diluted sulfuric acid and determine the effect of the pretreatments on the interaction between commercial enzymes for the production of fermentable sugars by enzymatic hydrolysis. Pretreatments with IL were more efficient in the removal of lignin than pretreatments with DSA. Cellulase and endo-1,4-xylanase had a higher contribution to the release of glucose and xylose for both pretreated samples. The highest values of glucose and xylose yields, 99.96% and 86.52%, respectively, were achieved with the enzymatic hydrolysis of the biomass pretreated with IL.