In South Africa, approximately 3 × 106 tons of sugarcane bagasse is produced annually by 14 factories located on the north coast of KwaZulu-Natal. It is one of the most readily available lignocellulosic materials for ethanol production through enzymatic saccharification and hydrolysis. Pre-treatment enables disruption of the naturally resistant structure of lignocellulosic biomass to make the cellulose accessible to hydrolysis for conversion to biofuels. In this study, pre-treatment of depithed bagasse and mill-run bagasse was done using acid (3% H2SO4 v/v) followed by alkali (4% NaOH w/v), and the pre-treated solid was subjected to enzymatic hydrolysis. The effects of different conditions for enzymatic saccharification such as enzyme dose, reaction time, and amount of surfactant were studied in detail. The pre-treated substrate (10% w/v) when hydrolysed using 30 FPU/gds/40 FPU/g dry substrate (gds) with 0.4% (v/v) Tween® 80 for 20 h resulted in 608 mg/gds (depithed bagasse) and 604 mg/gds (mill-run bagasse) total reducing sugars.