Purpose Induction of skin tissue regeneration and reduction of microbial infection at the wound site are crucial for burn wound treatment. In this study, human amniotic membrane (HAM) and titanium dioxide (TiO2) nanoparticle infused gel (HAM+TiO2) was developed to find a promising biomaterial for burn wound management. Methods HAM, TiO2, and HAM+TiO2 gels were prepared with carbopol 934, acrylic acid, propylparaben, and triethanolamine. Besides, physiochemical properties (pH, water absorption, swelling ratio, spreadability, etc.), antibacterial activity, and brine shrimp lethality tests were executed for the gels. The gels were applied to second-degree burns of Wistar rats for 20 days. Percent wound contracture, epithelialization period, and histological observations were carried out to evaluate the wound healing process. Results Formulated gels have shown antibacterial activity against S. aureus (ATCC 25923), P. aeruginosa (ATCC 27853), and E. coli (ATCC 25922). After 20 days of treatment, HAM+TiO2 significantly (P<0.0001) accelerated wound contraction (95.29 +/- 0.54%) compared to a control group. For complete healing, the epithelialization period was 21.0 +/- 1 day (P< 0.001). Histology analysis revealed the better wound healing potential for HAM+TiO2 in terms of epidermal regeneration, blood vessel formation, and collagen deposition with minimal scar formation. Conclusion Our findings demonstrated that the treatment of burn wounds with HAM+TiO2 significantly promoted wound healing in Wister rats by accelerating wound contraction, collagen synthesis, and preventing bacterial growth. Lay Summary and Future Works Burns have dreadful consequences for the affected patients, making them susceptible to wound infections. The application of HAM for wound healing is increasing because of its low immunogenicity, angiogenic, and antimicrobial properties. For burn wound treatment, TiO2 NPs have been well identified. In this research, TiO2 NP-incorporated HAM gel was prepared and we used this gel to experimental groups of Wister rats with an initial burn on their skin. In an in vivo study and histopathologic observation, better wound healing progress was demonstrated for HAM + TiO2. In the future, the study will be performed on large scale for the treatment of full-thickness burns and/or chronic wounds.