The enzymatic cocktail of cellulases is one of the most costly inputs affecting the economic viability of the biochemical route for biomass conversion into biofuels and other chemicals. Here, the influence of liquid hot water, dilute acid, alkali, and combined acid/alkali pretreatments on sugarcane bagasse (SCB) used for cellulase production was investigated by means of spectroscopic and imaging techniques. Chemical composition and structural characteristics, such as crystallinity (determined by X-ray diffraction), functional groups (Fourier transform infrared spectroscopy), and microstructure (scanning electron microscopy), were used to correlate SCB pretreatments with enzymatic biosynthesis by a strain of the filamentous fungus Aspergillus niger under solid-state fermentation. The combined acid/alkali pretreatment resulted in a SCB with higher cellulose content (86.7 %). However, the high crystallinity (74 %) of the resulting biomass was detrimental to microbial uptake and enzyme production. SCB pretreated with liquid hot water yielded the highest filter paper cellulase (FPase), carboxymethyl cellulase (CMCase), and xylanase activities (0.4, 14.9, and 26.1 U g-1, respectively). The results showed that a suitable pretreatment for SCB to be used as a substrate for cellulase production should avoid severe conditions in order to preserve amorphous cellulose and to enhance the physical properties that assist microbial access. © 2013 Springer Science+Business Media.