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Abstract Background Biodiesel is a type of renewable energy, an ideal substitute for petroleum diesel fuel. The present study concerns about optimization of culture conditions for biodiesel production by Penicillium commune NRC2016. Results The maximum lipid production from P. commune NRC2016 was investigated using basal liquid medium with initial pH 7.0, incubation temperature 20 °C, and after 5 days of incubation time at static condition. Six types of agro-industrial by-products (broken rice, rice straw, wheat bran, corn stalk, sweet sorghum, and bagasse) were separately used as components of semi-solid fermentation media. The highest lipid accumulation was recorded with sweet sorghum 99.1 mg/g as compared with the other by-products. Biodiesel obtained from P. commune NRC2016 was blended “B5” and the physical properties were determined and found to be as follows: density 0.8 g/ml, viscosity 2.1 mm2/s, flash point 77.0 °C, cloud point − 1.5 °C, iodine value 42.3 g I2/100 g, acid value 2.1 mg/g, pour point − 1.7 °C, and cetane number 47.8 min. Conclusions This work revealed the optimization of culture conditions for biodiesel production from Egyptian fungal strain P. commune NRC2016.