Starch-enriched brewery waste (SBW), an unexplored feedstock, was investigated as a nutritious low-cost source for the mixotrophic cultivation of Ettlia sp. YC001 for biodiesel production. Stirring, autoclaving, and sonication were assessed for the SBW, in conjunction with pH. Stirring at 55 °C was found to be the best, in terms of the effectiveness of starch hydrolysis and yeast disintegration as well as cost. The treated solutions were found to support the mixotrophic growth of microalgae: 20 g/L of glucose medium resulted in the highest biomass production of 9.26 g/L and one with 10 g/L of glucose showed the best lipid productivity of 244.2 mg/L/day. The unsaturated fatty acids increased in the resulting lipid and thus quality well suited for the transportation fuel. All these suggested that SBW, when treated properly, could indeed serve as a cheap feedstock for microalgae-based biodiesel production.