Collagen and its blends, collagen/polyvinyl alcohol (PVA) and collagen/chitosan/PVA, were made into nanofibers by electrospinning. The nanofibrous matrices were evaluated for their potential as skin substitutes. The addition of PVA to collagen increased the swelling ratio of the nanofibers, their Young's modulus, strain at break and ultimate tensile strength. The addition of chitosan to collagen/PVA reduced its swelling ratio and its strain at break, but increased the Young's modulus and ultimate tensile strength. Both PVA and chitosan stabilized the collagen fibers in an aqueous solution. The addition of PVA, but not chitosan, promoted initial fibroblast cell proliferation on the matrices. Compared to the skin substitute made of pure collagen, the substitutes with PVA and chitosan showed improved structural stability in aqueous solution, better tensile strength and similar or better biocompatibility in vitro.