In the quest for an ideal wound healing material, human amniotic membrane (AM), tilapia skin collagen (TSC), and Centella asiatica (CA) have been studied separately for their healing potential. In this study, we formulated AM, TSC, and CA gel and studied their competency and wound healing efficacy in vivo. Gel was formulated using AM, TSC, CA, Carbopol 934, acrylic acid, glycerine, and triethanolamine and physicochemical properties e.g.,pH, water absorption, swelling variation, and nuclear magnetic resonance (NMR) spectroscopy were determined. Biological properties were determined by skin irritation study, brine shrimp lethality, andantibacterial activity. Wound healing potential was determined by applying gel to second-degree burnsin vivoby observing wound contracture, epithelialization period, and histological features. The gel was non-lethal to brine shrimp and had anti-bacterial activity and showed no edema or erythema after 7 days of topical application. After 21 days of treatment, the AM + TSC + CA group significantly (P <
0.001) accelerated wound contraction (95.75 ± 0.44%)whereasthenegative control had the lowest healing rate (40.32 ± 2.11%). Wound contraction rates of AM + TSC and TSC + CA groups were (68.12 ± 1.46%) and (82.52 ± 1.74%) respectively.Epithelialization period for AM + TSC + CA was only 22.7days whereas AM, TSC, CA, AM + TSC, AM + CA, TSC + CA, positive control, and negative control needed 29.3, 30.7, 31.3, 27.3, 26, 26.6, 25.3 and 36.6 days respectively. Histological analysis showed better healing potential for AM + TSC + CA regarding epidermal regeneration, blood vessel formation, and collagen deposition. The gel was biocompatible and in vivostudies with Wistar rats exhibited better wound healing capabilities than individual components of the gel alone.