Surfactants are amphipathic molecules of petrochemical origin used in various industrial segments, mainly in the petroleum industry. However, they are toxic and produced from non-renewable sources. Biosurfactants are natural, biodegradable and less toxic molecules synthesized by bacteria, yeast, and filamentous fungi, however, their cost of production is still high. In the present study, agro-industrial by-products were used as renewable sources to evaluate their influence on the production of biosurfactant by Penicillium sclerotiorum UCP1361. A central composite rotated design (CCRD) 2² was performed to optimize the concentrations of milk whey and barley on the synthesis of biosurfactant. The filamentous fungus P. sclerotiorum was inoculated in the assays according to the experimental design and incubated in orbital agitation at 150 rpm, pH 5.0 for 96 h at 28 °C. Then, the cell-free metabolic liquid was obtained and the surface tension was measured. The emulsification index was also analyzed with different hydrophobic substrates. The dispersing capacity of the biosurfactant and its viscosity were tested on burnt engine oil. The extraction of the biomolecule was performed using chloroform and methanol as solvent system. The results showed that the biosurfactant reduced the surface tension from 72 to 27 mN/m and the emulsification index was 68% with burnt engine oil. The dispersing capacity detected was 33.15 cm2 and further reduced the oil viscosity from 279.6 to 48.5 cP. This study indicated that the biosurfactant produced by P. sclerotiorum may be promising as a bioremediation agent in oil spilled sites.