Gelatin nanofibers containing lipopeptide biosurfactant which had high surface activities and antimicrobial potential were fabricated by electrospinning technique to study their potentiality for biomedical applications. Operating voltage and concentration (w/w) of lipopeptide biosurfactant in 20% (w/v) gelatin solution were considered as the experimental variables at fixed flow rate, fabricating time, and distance between the needle tip and fiber collector. The diameter trend of the electrospun nanofibers increased with the increase of the operating voltage and the biosurfactant ratio. The contact angle between the nanofiber surface and water droplet was greater when compared with that of gelatin nanofiber without the biosurfactant. The percentage of the biosurfactant would affect the porosity of the obtained nanofiber mat. The most suitable gelatin nanofiber for wound healing and covering application under this study was obtained from 25% (w/w) lipopeptide biosurfactant in gelatin solution at 6 kV operating voltage. These nanofibers also showed reasonably antimicrobial property against E. coli and S. aureus. The novelty found in this study is that the percentage of water uptake which is one of the most important factors for wound healing and covering materials of the obtained electrospun nanofibers is much better than that of the commercially one used nowadays.