Biofuels produced from lignocellulosic biomass has been recognized as a potential sustainable source of energy. The process of converting lignocellulosic materials to bioethanol involves pretreatment, hydrolysis, and fermentation. Pretreatment is considered to be the crucial step because it governs the susceptibility of the biomass to the downstream hydrolysis and fermentation. However, the exact effects of pretreatment on the biomass are not thoroughly understood. This study provides information on the anatomical characteristics of Buddleja davidii substrate after ethanol organosolv pretreatment that can be responsible for efficient cellulose hydrolysis by cellulases. The pretreated wood was sectioned, stained, and observed under light microscopy. Improving our fundamental knowledge of pretreatment technologies will lead to significant advancements in this field to produce low-cost cellulosic ethanol. Results from the study showed that the ethanol organosolv pretreatment of B. davidii selectively removes lignin in the middle lamella, causes cell deformation, and cracks or breaks the cell wall, while retaining the crystallinity of cellulose. The removal of lignin from the middle lamella regions was the most pronounced microscopic observation and believed to be the major factor in efficient enzymatic hydrolysis, as the cellulose chains became more accessible to the enzymes. The enzymes were able to penetrate easily through the outer part of the cells where the middle lamella was absent.