The 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride-crosslinked collagen-hyaluronic acid (HA) matrices containing tobramycin or ciprofloxacin as an antibiotic agent were fabricated for the control of wound contamination and characterized with respect to morphology, mechanical strength, in vitro release, antibacterial activity and cytotoxicity. For the tobramycin loaded matrix, the antibacterial capacity increased with the drug loading. Tobramycin and ciprofloxacin loaded matrices maintained their antibacterial effects for over 96 and 48 h, respectively. However, cell viability testing revealed that 0.4 mg/ml of ciprofloxacin has a cytotoxic effect on fetal human dermal fibroblasts. The effects of the bilayered collagen-HA matrices containing tobramycin and growth factors were also evaluated using an in vivo full thickness dermal defect model. Though the tobramycin incorporated collagen-HA matrix had no significant effect on wound healing compared with the control, the tobramycin incorporated matrix containing basic fibroblast growth factor or platelet-derived growth factor significantly enhanced wound healing. This study demonstrates the potential efficacy of crosslinked collagen-HA matrix containing antibiotics and growth factors for defective skin tissue replacement and infection prevention.