Aims:The synergistic bio-activity between oleaginous yeast and microalga has been recognized, which would enhance lipid production as biodiesel feedstock. Nevertheless, yeast and microalga require different conditions for optimal growth. In this study, the locally isolated oleaginous yeast Rhodotorula toruloides and microalga Chaetoceros muelleri were cocultivated to enhance biomass and lipid production. Methodology and results:The growth characteristics of both yeast and microalga monocultures were initially determined prior to optimizing the co-cultivation conditions. The biomass and lipid productivity of the co-culture were investigated and compared to their monocultures. The results showed that R. toruloides grew actively within 3 days while C. muelleri exhibited more prolonged cultivation, up to 21 days. The co-cultivation could be carried out optimally using growth media at pH 6, light intensity of 15,000 lux and yeast/microalga ratio of 1:2, yielding the highest biomass productivity determined at 0.18 g/l/day and lipid production of 17%. The lipid productivity of the co-culture increased by 42% and 75% as compared to monocultures of yeast and microalga, respectively. Furthermore, the biomass productivity was also higher than the monoculture, about 1.2-fold for the yeast and 13-fold for the microalga. Conclusion, significance and impact of study:The findings revealed that co-cultivation of yeast and microalga is a viable technique for long-term microbial oil production.