Electrospun gelatin and poly-ε-caprolactone (PCL) nanofibers were prepared using needleless technology and their biocompatibility and therapeutic efficacy have been characterized in vitro in cell cultures and in an experimental model of a skin wound. Human dermal fibroblasts, keratinocytes and mesenchymal stem cells seeded on the nanofibers revealed that both nanofibers promoted cell adhesion and proliferation. The effect of nanofibers on wound healing was examined using a full thickness wound model in rats and compared with a standard control treatment with gauze. Significantly faster wound closure was found with gelatin after 5 and 10 days of treatment, but no enhancement with PCL nanofibers was observed. Histological analysis revealed enhanced epithelialisation, increased depth of granulation tissue and increased density of myofibroblasts in the wound area with gelatin nanofibers. The results show that gelatin nanofibers produced by needleless technology accelerate wound healing and may be suitable as a scaffold for cell transfer and skin regeneration.