As the number of consumers suffering from rough hands and dermatitis caused by the frequent daily use of synthetic surfactants increases, biocompatible materials are required. Biosurfactants (BSs), compounds excreted or produced by microbial cells, attract attention as cosmetic substrates suitable for human skin and the environment. This study evaluated the utility of the glycolipid-type sophorolipid (SL) produced by the non-pathogenic yeast Starmerella bombicola, as a BS. The cytotoxicity of open-chain acid type SL (SL acid) among SLs, is approximately 100–250 times less than that of commercially available surfactants in all cells. Therefore, SL acid is a promising surfactant with a high safety profile. In contrast, the critical micelle concentration (CMC) of SL acid, surfactin, and rhamnolipid were 1,000 mg/L, 16 mg/L, and 38 mg/L, respectively, indicating that SL acid has lower functionality than the other BSs. Finally, the safety range was analyzed for each BS to indicate practicality. The safety range, the concentration range where each BS can exhibit its function without cytotoxicity, was defined based on the lethal concentration 50 (LC₅₀)/CMC value. As a result, the safety range of SL acid is 3.9–4.4 times wilder than that of surfactin and rhamnolipids. Consequently, SL acid could be a promising BS with a wider safety range than other BSs, such as surfuctin and rhamnolipids.