In this study, nanofibrous membranes from biodegradable PLGA and collagen were fabricated to mimic natural extracellular matrix (ECM) and investigated on the morphology, conformational stability, cytotoxicity and cell attachment. The effect of PLGA or collagen nanofibrous membranes incorporating human dermal fibroblasts on wound healing was also evaluated using an in vivo full thickness dermal defect model. The circular dichroism measurements showed that electrospun collagen maintained its triple helix structure. In cytotoxicity test using L929 fibroblastic cells, electrospun PLGA or collagen nanofibrous membrane demonstrated no significant toxicity. It was also found that collagen and PLGA nanofibers favored cell attachment and proliferation. In vivo testing showed that the regeneration of dermis and epidermis treated with PLGA or collagen nanofibrous membrane incorporating dermal fibroblasts was accelerated. Therefore, this electrospun PLGA or collagen nanofibrous membrane might have potential efficacy in tissue engineering as skin substitutes.