Abstract: Pretreatment evaluations of biological sludge were performed to observe the enhancement of hydrogen production and repression of methanogenic activity using anaerobic sludge and sucrose as substrate. The treatments include heating (H), ozonation (O), drug (D) application using fluvastatin, Na2SO4(S) dosing and their combinations to make up for the total of 9 pretreatment methods. Heat treatment at 95°C for 45 min provided a complete methanogen repression and good enhancement of hydrogen production activity. The remaining pretreatments were imperfect either for repression or enhancement based on the application conditions and concentration ranges. The order of methane repression was: H>HO>HD>HS>O>D>OD>OS>S while hydrogen production was: HO>H>HS>OD>HD>OS>D>O>S. Heat treatment at the specified condition was considered sufficient and suitable for pretreatment of anaerobic sludge. The practical application was explored using Palm Oil Mill Effluent (POME) containing different amounts of solid content (15.830 g VSS L-1, namely LPOME and 21.445 g VSS L-1, namely HPOME) as substrate. Beside the difference in COD of nearly 16% which is higher for HPOME, a higher specific hydrogen production rate was obtained at 0.81 and 0.17 mL H2 g-1 COD h for LPOME and HPOME, respectively. It should also be noted that a prolonged lag-time during start-up was observed for HPOME as well as hydrogen suppression which may be associated with the solid content mainly lipids in wastewater. Thus, high solid content of wastewater may be of concerned for bio-hydrogen production.