Microbial biosurfactants have emerged as sustainable alternatives to promote plant growth and control phytopathogens in an eco-friendly manner. Their multifunctionality and low toxicity make them promising allies for modern agriculture. This study investigated the production of a biosurfactant by Penicillium rubens UCP 1213 using post-frying soybean oil (PFSO) and corn steep liquor (CSL) as low-cost substrates. A 2² full-factorial design was used to evaluate the effects of substrate concentration on biosurfactant production, assessed by the reduction in surface tension. The best result was achieved in a medium containing 3% PFSO and 4% CSL, resulting in a surface tension of 26.8 mN/m. A reduction in the interfacial tension against n-hexadecane was also verified (1.8 mN/m). The biosurfactant was isolated with ethanol at 70% (2,40 g/L) and exhibited a critical micelle concentration (CMC) of 500 mg/L, indicating its effectiveness. In vitro assays demonstrated that the biosurfactant significantly enhanced the germination and vigor of onion seeds (Allium cepa L.) and inhibited the growth of phytopathogenic fungi Curvularia lunata and Fusarium oxysporum. These findings reinforce this biosurfactant's potential as a biostimulant and biocontrol agent. Therefore, the production of biosurfactants from agro-industrial waste represents a promising strategy for more sustainable agricultural practices in onion cultivation.