Effects of sodium and magnesium supplement on biolipid production simultaneously with wastewater treatment by Rhodosporidium toruloides were investigated using synthetic wastewater with the composition similar to the mixture of real distillery and domestic wastewater. When Mg²⁺ was supplemented as MgCl₂ at 200 mg/L, both lipid production and wastewater treatment were enhanced. The biomass and lipid production increased from 6.48 ± 0.69 and 0.96 ± 0.13 g/L to 7.78 ± 0.58 and 1.35 ± 0.02 g/L, respectively. The removal efficiencies for chemical oxygen demand (COD) and total phosphorus (TP) also increased from 72.09 ± 3.32 and 19.57 ± 2.51% to 79.20 ± 1.13 and 23.19 ± 3.07%, respectively. These increases were considered mainly due to the important role of Mg²⁺ in lipid synthesis, phosphate transfer, and carbohydrate metabolism. In comparison to Mg²⁺, the effect of Na⁺ depended on the supplement form. When 200 mg/L Na⁺ were provided as NaCl, maximum lipid production was 2.66 ± 0.11 g/L while only 1.98 ± 0.63 g/L when Na ⁺ provided as Na₂SO₄. For a better lipid production, the optimal concentrations of both Mg²⁺ and Na⁺ were considered around 200 mg/L. Both sodium and magnesium supplement did not show any obvious negative impact on biodiesel quality. The estimated values of important parameters of biodiesel with sodium or magnesium supplement met ASTM D6751 standard