Batch production of biohydrogen from cassava wastewater pretreated with (i) sonication, (ii) OPTIMASH BG® (enzyme), and (iii) α-amylase (enzyme) were investigated using anaerobic seed sludge subjected to heat pretreatment at 105 °C for 90 min. Hydrogen yield at pH 7.0 for cassava wastewater pretreated with sonication for 45 min using anaerobic seed sludge was 0.913 mol H2/g COD. Results from pretreatment with OPTIMASH BG® at 0.20% and pH 7 showed a hydrogen yield of 4.24 mol H2/g COD. Superior results were obtained when the wastewater was pretreated with α-amylase at 0.20% at pH 7 with a hydrogen yield of 5.02 mol H2/g COD. In all cases, no methane production was observed when using heat-treated sludge as seed inoculum. Percentage COD removal was found to be highest (60%) using α-amylase as pretreatment followed by OPTIMASH BG® at 54% and sonication (40% reduction rate). Results further suggested that cassava wastewater is one of the potential sources of renewable biomass to produce hydrogen.