With increasing energy requirements and limited fuel resources, finding cheap renewable sources is essential. The objective of our current economic study is to enhance biodiesel production from the Penicillium commune, NRC 2016. Four mutagenic agents were used to improve the lipid production from P. commune NRC 2016: gamma radiation, ethyl methanesulfonate (EMS), ethidium bromide (Et Br), and sodium azide (NaN3). The Inter-Simple-Simplece Repeats molecular (ISSR) marker was used to compare the wild type of P. commune NRC 2016 and the resulting mutants. The results showed a major difference between the wild type and its mutants. Response surface methodology was used to optimize the culture conditions for lipid production by P. commune NRC 2016, and the induced mutants used hydrolysate that was produced from bagasse using Bacillus cereus 3SME. The maximum lipid content (g/l) for P. commune NRC 2016, P. commune NRC 2016–1, P. commune NRC 2016–2, and P. commune NRC 2016–3 were 2.01, 2.55, 1.71, and 2.27, respectively. Gas chromatographic analysis was used for biodiesel produced from P. commune NRC 2016 wild type, and its mutants were mainly C16–C18, which is suitable for transesterification. The physical properties, such as density, viscosity, cloud point, pour point, and cetane number, of the biodiesel from P. commune NRC 2016 and its mutants were similar to those of standard biodiesel and could be applied on a large scale. The biodiesels produced through P. commune NRC 2016 and its mutants can be utilized on a broad scale as eco-friendly products obtained from a low-cost carbon source.