This study aimed to develop a novel wound dressing composed of hyaluronic acid (HA) spongy sheet containing bioactive components. The wound dressing prepared by the freeze-drying method has a two-layered structure: an upper layer composed of cross-linked high-molecular-weight HA (HMW-HA) and a lower layer composed of low-molecular-weight HA (LMW-HA) containing arginine (Arg), magnesium ascorbyl phosphate (vitamin C derivative: VC), and epidermal growth factor (EGF) (referred to as EGF-dressing). A wound dressing containing only Arg and VC was prepared in a similar manner (referred to as EGF-free-dressing). The potential of each wound dressing was evaluated in animal tests using Sprague Dawley (SD) rats and diabetic mice. In the first experiment, each wound dressing was applied to a full-thickness skin defect in the abdominal region of SD rats. Wound conditions after 1 week and 2 weeks of treatment were evaluated based on macroscopic and histological appearance. A commercially available non-woven alginate wound dressing (Alg-dressing) was used in a control group. Both EGF-free-dressing and EGF-dressing decreased wound size and promoted granulation tissue formation associated with angiogenesis more effectively when compared with Alg-dressing. In particular, EGF-dressing promoted re-epithelialization. In the second experiment, each wound dressing was applied to a full-thickness skin defect in the dorsal region of diabetic mice. Wound conditions after 1 week and 2 weeks of treatment were evaluated based on macroscopic and histological appearance. A commercially available Alg-dressing was used in a control group. Both EGF-free-dressing and EGF-dressing decreased wound size and promoted granulation tissue formation associated with angiogenesis more effectively when compared with Alg-dressing. These findings indicate that EGF-free-dressing and EGF-dressing have the potential for more effective wound healing when compared with Alg-dressing. In particular, EGF-dressing has a higher potential for wound healing when compared with EGF-free-dressing.