Lipopeptides from Bacillus subtilis represent a class of biosurfactants with increasing therapeutic and biotechnological interests. In this study, we added crude extract of lipopeptides into a gelatin solution to examine the effect of these crude lipopeptides using electrospinning. The addition of crude lipopeptides to a gelatin solution affected the solution viscosity and conductivity and resulted in a decrease in nanofiber diameter when the solution was electrospun to produce nanofibers. Crosslinking of nanofibers using saturated glutaraldehyde for 10 and 30 min improved the mechanical properties and water retention capacity of the mats formed from the nanofibers. Furthermore, the lipopeptides-loaded nanofiber mats showed antibacterial activity toward Staphylococcus aureus ATCC 6538P.