Biosurfactants have attracted considerable attention because of their lower toxicity, biocompatibility, and effectiveness over chemical surfactants. The use of renewable sources and the concept of sustainable production for such biomolecules supports the increased demand for eco-friendly products. Herein, the present study investigated corncobs (CC) and sunflower stalks (SS) as substitutes for conventional substrates in submerged fermentation with B. subtilis. The agro-industrial residues were submitted to an alkaline pretreatment to obtain hydrolysates rich in hemicelluloses, whose concentrations were determined at 48.8% and 65.7% for corncob and sunflower stalk liquors, respectively. The influence of different concentrations of glucose (0, 2.5, and 5%) and liquor (0, 20%, and 40%) were evaluated according to cell concentration, surface tension reduction rate (STRR), and emulsification index (EI24). Biosurfactants obtained with the hemicellulose liquor of sunflower stalk showed the highest cell concentration (4.57 g/L) and STRR (58.07%), whereas the maximum values of EI24 (56.90% in hexane, 65.63% in toluene, and 64.86% in kerosene) were achieved by using corncob liquor. All top results were observed at 2.5% glucose, 20% liquor (CC or SS), and 1% mineral salts. Notably, excess glucose or liquor (CC or SS) negatively affected cell growth and biosurfactant performance. The results indicated the potential of corncobs and sunflower stalks as low-cost substrates to produce a high added-value biosurfactant with promising tensoative and emulsifying properties.