HEREIN, we assessed oleaginous fungi for biodiesel production. We isolated 10 fungal strains from two petroleum-polluted soil samples and screened their ability to accumulate lipids. Aspergillus terreus, A. niger, and A. flavus were found to be the most potent lipid producers. All isolates were identified at the species level by morphological (macroscopic and microscopic) examination and molecularly confirmed by performing 18S rRNA sequencing. Furthermore, we optimized culture conditions to improve lipid accumulation. Aspergillus terreus, the most potent lipid producer, showed the highest lipid production (38.33%) in the presence of 50g/L sucrose and 0.5 g/L ammonium nitrate (initial pH, 6) after seven days of incubation under static conditions. Gas chromatography was performed for fatty acid analysis after lipid transesterification. Fatty acid methyl/ethyl ester profile indicated that saturated fatty acids were more common than polyunsaturated fatty acids. The total concentration of fatty acids was 107.98, 38.29, and 37.48mg/100g of lipids accumulated by A. terreus, A. niger, and A. flavus, respectively. Gas chromatographic analysis of A. terreus lipids indicated that oleic acid (C18:1, 18.51%) was the most abundant, followed by stearic acid (C18:0, 15.91%) and myristic acid (C14:0, 14.64%). The fatty acid profile of A. terreus confirmed it to be a promising new commercial biodiesel feedstock.