Biodiesel, known as fatty acid methyl esters (FAMEs), is a sustainable and eco-friendly substitute for Petro-diesel. This study investigates biodiesel synthesis from oil derived from oleaginous fungi, specifically, Fusarium verticillioides AUMC 16026, using Rhizopus arrhizus AUMC 16025 and Penicillium crustosum whole-cell lipase as a biocatalyst. This research marks the first record of using P. crustosum whole-cell lipase as a biocatalyst for biodiesel synthesis. Twenty-two fungi were isolated and screened for their ability to accumulate lipids and produce lipase. F. verticillioides AUMC 16026, the most potent lipid producer, showed the highest lipid production when grown in the presence of sucrose and sodium nitrate (initial pH, 4) for 3 days at 30°C under static conditions. R. arrhizus AUMC 16025 has the highest lipase activity (4.67 U/g), followed by P. crustosum (3.67 U/g). Therefore, they were chosen to perform fungal oil transesterification process. Gas chromatography was utilized for analyzing produced FAMEs. Analysis of biodiesel produced from F. verticillioides AUMC 16026 oil transesterified using R. arrhizus AUMC 16025 whole-cell lipase revealed that linoleic acid (27.28%) and oleic acid (25.46%) were the most abundant methyl esters. Meanwhile, that produced using P. crustosum whole-cell lipase showed oleic acid (23.53%) and palmitoleic acid (21.28%) were the most abundant, followed by linoleic acid (18.88%) and palmitic acid (11.84%). The biodiesel produced by both lipases has a quality that is similar to each other and to biodiesel made from vegetable and waste frying oils. This indicates that F. verticillioides AUMC 16026 oil is a good feedstock for biodiesel production.