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Background: Climate change is still a problem we face that has an impact on us every day even as our world develops technologically. Energy has been and will continue to be a key engine of economic growth. The greenhouse gas emissions (GHG) from biodiesel are less than those of fossil fuels. The objective was to improve biodiesel production from a fungus called Fusarium oxysporum NRC 2017 using the bagasse hydrolysate of Bacillus cereus 3SME as a cheap carbon source. Results: To enhance lipid accumulation in F. oxysporum NRC 2017, four mutagenic agents were used: gamma radiation (Ɣ ray); ethidium bromide (Et Br); ethyl methane sulfonate (EMS); and sodium azide (NaN3). Using the Inter-Simple Sequence Repeats (ISSR) as a molecular biomarker, the findings demonstrated a significant difference between the F. oxysporum NRC 2017 wild type and induced mutants examined. The culture conditions were optimized for F. oxysporum NRC 2017 and F. oxysporum NRC 2017-1, F. oxysporum NRC 2017-2, and F. oxysporum NRC 2017-3 using the response surface methodology (RSM) and the maximum lipid content (g/l) was 2.40, 3.47, 3.81, and 2.63, respectively. According to gas chromatography (GC) analysis, the biodiesel compositions produced from the F. oxysporum NRC 2017 wild type and its mutants exhibited generally C16-C18 and were suitable for biodiesel synthesis. F. oxysporum NRC 2017 and mutant biodiesels indicated physical features that were close to those of standard biodiesel. Conclusion: The biodiesels produced from the F. oxysporum NRC 2017 and its mutants could be used for large scale as eco-friendly products from a cheap carbon source.