This well-known important effect of particle size in solid-state fermentations was also shown to be valid in three-phasic cultivations employing different particle sizes of sugarcane bagasse in shake flask cultivations. It was observed that as the sugarcane bagasse particle size was increased up to 2 mm, the solid substrate acted as a suitable support for fungal growth, probably due to increased interaction between the fungus and the inducer substrate and, consequently, increased enzyme production. However, the increase of sugarcane bagasse particle size was not beneficial in cultivations using particle sizes larger than 2 mm, because the intense fungal growth resulted in dense fungus-substrate agglomerates (for both the conventional and the sequential fermentation methods), which compromised the homogeneity of the cultivation broth. Cultivations with particle sizes from 2.0 to 2.4 mm were therefore discarded.