The effect of four selected variables on Chemical Oxygen Demand (COD) removal and H2 production by anaerobic mixed cultures from tapioca wastewater in batch mode (viz. ferrous sulphate (FeSO4), initial pH, sodium bicarbonate (NaHCO3) and nutrient solution with two inoculums (3,750 mgVSS/L and 7,500 mgVSS/L) were sought. Identification and screening of significant variables were conducted using the Plackett-Burman Design. An independent sample t-test was applied using 12 trials to evaluate inoculums content to determine the optimum level of the main variables and inoculum content at the steepest ascent. FeSO4 and initial pH both had a statistically significant (P<0.05) influence on COD removal and H2 production. COD removal and H2 production was greater at 7,500 mgVSS/L inoculums content than at 3,750 mgVSS/L (P< 0.05). An initial pH of 10 and FeSO4 at 2.5 g/L yielded the maximum H2 production potential (443.37 mL H2/L) and COD removal (61.54 %).