The objective of this study was to investigate structural changes and lignin redistribution in Eucalyptus globulus pre-treated by steam explosion under different degrees of severity (S-0), in order to evaluate their effect on cellulose accessibility by enzymatic hydrolysis. Approximately 87.7% to 98.5% of original glucans were retained in the pre-treated material. Glucose yields after the enzymatic hydrolysis of pre-treated material improved from 19.4% to 85.1% when S-0 was increased from 8.53 to 10.42. One of the main reasons for the increase in glucose yield was the redistribution of lignin as micro-particles were deposited on the surface and interior of the fibre cell wall. This information was confirmed by laser scanning confocal fluorescence and FT-IR imaging; these microscopic techniques show changes in the physical and chemical characteristics of pre-treated fibres. In addition, the results allowed the construction of an explanatory model for microscale understanding of the enzymatic accessibility mechanism in the pre-treated lignocellulose.