Wastewater treatment using the activated sludge method requires a large amount of electricity for aeration. Therefore, wastewater treatment using co-culture systems of microalgae and heterotrophic microorganisms, which do not require aeration, has attracted attention as an energy-saving alternative to the method. In this study, we investigated different combinations of microalgae and heterotrophic microorganisms to improve the efficiency of wastewater treatment. Three types of microalgae and five heterotrophic microorganisms were used in combination for wastewater treatment. The combination of Chlamydomonas reinhardtii NIES-2238 and Saccharomyces cerevisiae SH-4 showed the highest wastewater treatment efficiency. Using this combination for artificial wastewater treatment, the removal rates of total organic carbon, PO43−, and NH4+ reached 80%, 93%, and 63%, respectively, after 18 h of treatment. To the best of our knowledge, this is the first study to show that a combination of green algae and yeast improves the efficiency of wastewater treatment. Transcriptome analysis revealed that the combined wastewater treatment altered the expression of 1371 and 692 genes in C. reinhardtii and S. cerevisiae, respectively. One of the main reasons for the improved wastewater treatment performance of the combination of green algae and yeast was the increased expression of genes related to the uptake of phosphate and ammonium ions in the green algae. As both the green algae C. reinhardtii and the yeast S. cerevisiae are highly safe microorganisms, the establishment of their effective combination for wastewater treatment is highly significant.Key points• Combination of various microalgae and heterotrophic microorganisms was tested• Combination of green algae and yeast showed the highest efficiency• This is the first report that this combination is effective for wastewater treatment