The development of novel local therapies for thermal burns (TB) and their pathogenetic rationale are a pressing challenge. Melatonin (MT) is an endogenous factor of hemostasis regulation with pleiotropic potential. The aim of this study was to assess some parameters of tissue regeneration, the functional state of mast cells and the levels of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) in the experimentally induced TB treated with the original MT-enriched dermal film (DF). A second-degree burn (3.5% of the total body surface area) was modelled by exposing a patch of skin to hot water. Applications of 12 cm² DF enriched with 5 mg/g MT were performed every day for 5 days. The following parameters were calculated: the wound area, the rate of wound epithelization, the number of MC in the wound, the intensity of degranulation, and the levels of MMP-9 and VEGF expression.  Over the course of treatment, the absolute wound area shrank by 35%, its epithelization rate increased, the number of MC rose, their functional state changed, and the expression of ММР-9 and VEGF increased. A negative correlation was established between the wound area and the expression of ММР-9 and VEGF, as well as between the wound area and the degranulation coefficient. Applications of MT-enriched DF resulted in the reduction of the wound area, higher epithelization rate, an increase in the total MC count and degranulation intensity on days 5 and 10; it also led to a reduction in the total MC count and a loss in degranulation intensity on day 20 (166.87 (154.95; 178.78) un/mm² vs. 464.84 (452.92; 476.76) un/mm²) in the group of intact animals), an increase in MMP-9 expression on day 5 (14.20 (11.30; 18.10) vs. 3.30 (2.20; 4.40) in the intact group), an increase in VEGF expression on days 5 and 10 (33.00 (30.20; 34.90) vs 25.40 (22.20; 29.30) in the intact group), and a reduction in MMP-9 expression on days 10 and 20 after thermal injury.