Wound dressing materials must have a large pore size enough to provide sufficient room for cellular ingrowth. Meanwhile, for providing appropriate minimal water permeability, a smaller pore diameter is required for preventing the wound from drying. Therefore, these requirements must also be satisfied by skin substitutes prepared by tissue engineering. In these regards, We fabricated the bi-layered collagen-hyaluronic acid (HA) matrix which consisted of a dense collagen membrane and a porous collagen-HA matrix by freeze drying technique and cross-linked by EDC (1-ethyl-3(3-dimethyl aminopropyl) carbodiimide) to achieve a mechanical stability of the matrix. Tobramycin as an antibiotic agent, platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) were incorporated into the bi-layered matrices in order to prevent wound contamination and to enhance the rate of wound healing. The aim of the study was to develop the bi-layered collagen-HA matrices containing antibiotics and growth factors and to evaluate the effect of the matrices on the wound healing by using an in vivo full thickness dermal defect model in guinea pigs. When comparing the effect of bi-layered collagen-HA matrix on reduction of wound size in guinea pigs with a polyurethane matrix demonstrated significantly higher reduction of wound size on 7, 14 and 21 days after application. When comparing the effect of an antibiotic loaded collagen-HA matrix and an antibiotic, PDGF loaded collagen-HA matrix on reduction of wound size in guinea pigs with polyurethane matrix demonstrated significantly higher reduction of wound size on 3, 7 and 14 days after application. When comparing the effect of an antibiotic, bFGF loaded collagen-HA matrix on reduction of wound size in guinea pigs with polyurethane matrix demonstrated significantly higher reduction of wound size on 3, 7, 14 and 21 days after application. These results suggested that the bi-layered collagen-HA matrices containing antibiotics and growth factors synergistically improved wound healing.