Recently, the mass scale production of fucoxanthin and fatty acid from microalgae has increased. However, a new and low cost cultivation strategy is needed to enhance the production. This work investigated the potency of mixed diatom and green algae isolated from Morosari Bay, Java Sea, Indonesia, to enhance the production of fucoxanthin and lipid by employing mixotrophic condition. A four stepwise experiment was done to study the environmental and nutritional factors, as well as to optimize the selected parameters by employing Box Behnken response surface methodology. The mixed algae was found to grow well on palm oil mill effluent (POME). Changing condition from autotrophic to mixotrophic decreased the pigment content. The highest fucoxanthin concentration (6 mg L−1) was found on the glycerol addition compared to other organic carbon sources. The optimal condition to produce 160 mg L−1 d−1 biomass and 60 mg L−1 d−1 lipid was found on 30% POME, supplemented with 3 g L−1 glycerol and 200 mg L−1 urea at 30 °C. Mono unsaturated fatty acid (MUFA) was the most dominant fatty acid class found in the mixed algae.