Biosurfactants (BS) are amphiphilic molecules, more biocompatible and biodegradable than their chemical homologous, used to reduce water surface tension and stabilize non-aqueous soluble active principles. BS can improve drug solubility reducing phase separation and aggregation. Thus, BS can be good candidates for combination with poorly water-soluble Gramicidin S (GR-S) enhancing its antimicrobial activity in aqueous environment against Staphylococcus aureus (S. aureus) and reducing the potential toxicity for human skin natural microbiome like Staphylococcus epidermidis (S. epidermidis), as ethanol is commonly used solvent in pharmaceutical formulations. In this study, two formulations containing GR-S dissolved in the aqueous solutions (1 g/L) of cell-bound or extracellular BS, coming from corn steep water (CSW), were prepared. Minimum inhibitory concentrations (MIC) against S. aureus and S. epidermidis were assayed after 24 h using an ethanolic solution of commercial GR-S as a reference. Results showed that GR-S formulations in BS solutions presented similar MIC values against S. aureus compared to the reference GR-S in ethanol. Moreover, GR-S formulation in extracellular BS keeps the selectivity toward S. aureus in comparison with S. epidermidis. In conclusion, aqueous solutions of cell-bound and extracellular biosurfactants derived from CSW can replace ethanol and/or synthetic surfactants in pharmaceutical formulations containing GR-S as an active ingredient.