Cellobiose lipids (CBLs) are a class of glycolipid biosurfactants produced by various fungal strains. These compounds have gained significant interest due to their surface-active and antifungal properties, which are comparable to traditional synthetic surfactants and antimicrobials. Despite their potential applicability in various cosmetic, pharmaceutical, and agricultural formulations, significantly less research has been focused on their production and purification in comparison to other glycolipid biosurfactants, such as mannosylerythritol lipids (MELs) and sophorolipids. Hence, this work proposes the development of a bioprocess that involves the microbial production and high-level chromatographic purification of CBLs from a submerged culture of Ustilago maydis DSM 4500. After a highly purified CBL product was obtained, the factors affecting the production of this glycolipid were investigated. It was demonstrated that U. maydis DSM 4500 produces a specific structural variant of CBLs at a concentration of 1.36 g/L on an optimized the growth medium. Also, it was established that when the C/N ratio was decreased, the CBL titer increased by 2.3-fold. Furthermore, supplementing the culture with ZnSO