Recent advancements in wound care have explored the use of biological dressings, including fish skin, due to its rich collagen content and bioactive components that promote healing. This study aimed to evaluate the effectiveness of fresh and lyophilized Nile tilapia skin in enhancing full thickness skin wound healing in donkeys. Nile tilapia skin was collected, thoroughly washed, lyophilized, and sterilized. Five female donkeys were used in the study, each receiving three full-thickness skin wounds (2 cm x 2 cm) on each side of the back after aseptic preparation and local anesthesia, resulting in a total of six wounds per animal. The wounds were assigned to three groups: control (treated with saline), fresh fish skin, and lyophilized fish skin. Macroscopic wound assessment was performed and skin samples were collected on days 14 and 28 for histological examination using hematoxylin and eosin (H&E) and Crossman’s trichrome staining. Results revealed that treatment with lyophilized fish skin significantly accelerated wound contraction and epithelialization compared to the control and fresh fish skin-treated groups. On day 14, wound contraction rates were 43.57% ± 0.87 for lyophilized fish skin, 41.32% ± 0.26 for fresh fish skin, and 32.48% ± 0.39 for the control. By day 28, contraction rates increased to 74.37% ± 0.77, 66.92% ± 0.31, and 56.88% ± 0.73, respectively. Histological analysis showed enhanced collagen deposition and angiogenesis in the lyophilized fish skin group. In conclusion, lyophilized Nile tilapia skin is a promising and cost-effective biomaterial for enhancing wound healing, offering a practical solution for field veterinarians in low-resource settings.