Various pre-treatment methods including heat have been conducted on the mixed inoculums to enrich hydrogen producing bacteria (HPB). This study investigated the use of heat as pre-treatment method for the anaerobic sludge at 105oC ranging from 45, 60, 90 and 120 minutes to determine the optimum time to suppress methanogenic activity and enhance hydrogen production using artificial wastewater. Optimum time of heating the inoculum obtained from the experiment was applied to the sugar mill wastewater as substrate. Sugarcane mill wastewater was also diluted to 50% to see the effect on H2 production. Results of batch experiments showed that heat treatment at 105oC regardless of time duration of heating showed no activity for methanogenic bacteria. There is an increasing trend of hydrogen production with the increase in heating time at 105oC regardless of sucrose loading (10g/L and 20g/L) with the highest value recorded at 120 minutes (3.24mol H2/gCOD for 10g/L and 4.26mol H2/gCOD for 20g/L). Results for sugarcane mill wastewater revealed an increasing trend of H2 production with the highest value of 2.38mol H2/gCOD observed for 100% substrate loading (without dilution) and 1.75mol H2/gCOD for 50% dilution indicating a suitable source for clean energy production.