Volatile Fatty Acids (VFAs) have become a subject of significant interest recently, primarily because they can serve as a carbon source for microorganisms that produce bioactive compounds, biodegradable materials, and energy. VFAs can be produced from various waste streams via anaerobic fermentation (AF) in a cost-effective manner, providing a potential avenue for producing biofuels and biochemicals while reducing waste volume. In this study, different VFAs and glycerin individually or in combination were used as the sole carbon source in the mixotrophic cultivation of oil-rich filamentous Tribonema minus. Using different VFAs or glycerin individually caused pH fluctuation in the culture, which led to the accumulation of free ammonia, which produced protons, acidifying the environment and inhibiting microalgae growth. To minimize pH fluctuation, different proportions of organic carbon sources were combined. Under combined organic carbon sources, the growth and photosynthetic performance of T. minus were all higher than those of the single carbon source groups. When Cacetic acid:Cbutyric acid:Cglycerin was set to 0.5:0.5:1, pH in the culture remained close to neutrality, and the removal of NH4+-N and carbon reached the maximum of 81.17 and 97.79%, respectively. The microalgal biomass and lipid productivity reached the highest of 223.80 and 108.16 mg L−1 d−1, respectively. Under the optimized carbon source condition, the fatty acids and biodiesel-related qualities of T. minus were improved. This study provided a reference for the precise design of carbon sources to maximize the resource utilization of butyric acid-type AF effluent by oil-rich T. minus. These conditions also can be achieved on a large scale by adding glycerin wastes to butyric acid-type AF effluent, mainly composed of acetic and butyric acids to get the recommended ratio for microalgae cultivation.