Mixotrophic microalgal cultivation can utilize CO₂ and organic carbon sources. This study optimized the cultivation nitrogen source (peptone, urea, yeast extract, NH₄Cl, (NH₄)₂SO₄, NH₄NO₃, NaNO₃ and KNO₃), carbon combination (glucose, glycerol, sucrose), (NH₄)₂SO₄ nitrogen source and pH (6–11) in a local microalgae species with three characteristics (high pH-resistant, high growth rate and high lipid content). Chlorella sp. G3H3-1-2 biomass production and lipid accumulation were estimated using the fatty acid methyl esters (FAMEs) concentration. The Chlorella sp. G3H3-1-2 FAME content was strongly influenced by the carbon, nitrogen sources and pH variations. The pH ranged from 6.0 to 8.0, which produced the highest specific growth rate of 1.22 day⁻¹ for Chlorella sp. G3H3-1-2 while using glucose as the single carbon source. However, the highest total FAMEs content of 59% in the Chlorella sp. G3H3-1-2 biomass of 1.75 g/L was obtained while using the combination of 1 g-glucose/L as the carbon source and 0.2 g-(NH₄)₂SO₄/L as the nitrogen source at the high pH value of 10.