In this work, laccase biosynthesis of two microorganisms, Trametes versicolor TISTR 3224 and Bacillus sp. TISTR 908 isolated in Thailand, was investigated using sugarcane bagasse (SCB) as substrate. Two-stage biological/physico-chemical pretreatment of SCB on delignification and saccharification yield was studied. A two-level full factorial design was applied and 3 factors influencing delignification and saccharification processes of SCB were studied including C:N ratio (10:1 to 20:1), temperature (100 to 140°C), and alkali concentration (0 to 5% w/w NaOH). It was found that during biological pretreatment of SCB, a greater amount of laccase was produced from T. versicolor in the early stage of growth compared with Bacillus sp. Nitrogen supplement enhanced laccase biosynthesis of T. versicolor. By contrast, Bacillus sp. required a smaller amount of nitrogen source to produce laccase. Biological treated bagasse was subsequently subjected to a physico-chemical treatment. The results showed that the highest xylose and glucose yield of 51.97% w/w based on carbohydrate content was obtained from T. versicolor cultivation at a C:N ratio of 20:1, and consecutively treated in 5% w/w NaOH solution at 140°C for 1 h. Bacterial/alkali and alkali pretreatment yielded xylose and glucose in smaller degrees compared with fungal/alkali pretreatment. T. versicolor preferentially degraded lignin in sugarcane bagasse relative to cellulose and hemicelluloses constituents, while Bacillus sp. simultaneously attacked both lignin and carbohydrate moieties, as indicated by analysis of relative FT-IR intensities ratios of pretreated and untreated sugarcane bagasse.