Improper lignocellulosic wastes management causes severe environmental pollution and health damage. Conversion of such wastes particularly sugarcane bagasse (SCB) onto bioenergy is a sustainable approach due to a continuous depletion of conventional biofuels. The delignification of SCB is necessary to proceed for bio-genic H₂ productivity by anaerobic bacteria. The effect of autoclaving, pre-acidification/autoclaving and pre-alkalization/autoclaving of SCB on glucose recovery and subsequently H₂ productivity by dark fermentation was comprehensively investigated. Pre-acidified SCB with 1% H₂SO₄ (v/v) provided H₂ productivity of 8.5 ± 0.14 L/kg SCB and maximum H₂ production rate (R_m) of 105.9 ± 8.3 mL/h. Those values were dropped to 2.7 ± 0.13 L/kg SCB and 58.3 ± 12.9 mL/h for fermentation of delignified SCB with 2% H₂SO₄. This was linked to high levels of total phenolic compounds (1775.3 ± 212 mg/L) in the feedstock. Better H₂ productivity of 13.9 ± 0.58 L/kg SCB and R_m of 133.9 ± 3.6 mL/h was achieved from fermentation of pre-alkalized SCB with 1%KOH (v/v). 256.8 ± 9.8 U/100 mL of α-amylase, 165.7 ± 7.6 U/100 mL of xylanase, 232.8 ± 6.1 U/100 mL of CM-Cellulase, 176.5 ± 5.0 U/100 mL of polyglacturanase and 0.702 ± 0.013 mg M B. reduced/min. of hydrogenase enzyme was accounted for the batches supplied with delignified SCB by KOH. The Clostridium and Bacillus spp. was dominance and prevalence resulting a higher H₂ productivity and yield. A novel strain of Archea and alpha proteobacterium were also identified and detected.