Sophorolipids are among the best-positioned microbial biosurfactants to reach large-scale industrial production and application. However, the structural variety of sophorolipids offered by wild-type strains is rather limited, requiring their efficient modification to expand the application areas of sophorolipids. A combination of genetic engineering and green chemical modification via ozonolysis was applied in this work to produce key precursors useful in the development of a library of sophorolipid derivatives. Uniform symmetrical α,ω-bola sophorosides, produced by a novel strain of Starmerella bombicola, were investigated as substrates for the first time to generate 100% ω-C9 sophorosides (key precursors in the development of a sophorolipid library) via ozonolysis in water. Ozonolysis yielded a mixture of C9:0 ω-sophoroside aldehydes and C9:0 ω-sophorolipid acids. The selectivity toward the C9:0 ω-sophoroside aldehyde was increased using catalase, limiting the overoxidation of the aldehyde by the in situ formed H2O2. The C9:0 ω-sophorolipid acid could be produced selectively by extending the ozonolysis time. Moreover, using water as the solvent during ozonolysis proved to be beneficial in suppressing the formation of ozonides, therefore eliminating the need to perform a reductive or oxidative workup. Consequently, an efficient, safe, and scalable route has been established for the production of key sophoroside precursors.