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.