Background <p><i>Citrus japonica (C. japonica</i>), known as Kumquat, is small-size edible fruit native to South Asian regions and reported for its anti-inflammatory and antioxidant properties. This manuscript aimed to investigate LC-HRESIMS-based metabolomic profiling and wound healing properties of <i>C. japonica</i> fruit extract hydrogel formulation.</p> Methods <p>The metabolic profiling of the fruit extract was investigated using LC-HRESIMS. The extract was formulated in hydrogel preparation and examined for in vivo wound healing activity. Twenty rats were divided into four different groups (n = 5); the negative control group did not undergo any wound creation; the positive control group was wounded and did not receive daily application of a topical formulation based on the wound area; the reference group consisted of wounded rats treated with MEBO® ointment applied to the wound area. The remaining group of rats were wounded and received topical preparations of <i>C. japonica</i> with variations in the size of the applicator head. The effects of <i>C. japonica</i> fruit extract were examined on all phases of wound healing via assessment of the expression levels of key markers such as TNF-α, VEGF, PDGF, and TIMP-2. Besides, morphological and histopathological studies were conducted.</p> Results <p>The findings reveal that treatment with <i>C. japonica</i> extract expedites wound healing, showing comparable effectiveness to standard treatments like MEBO®. Understanding these molecular markers provides valuable insights for designing precise treatments that enhance the wound healing process and minimize complications in clinical settings. The LC-HRESIMS revealed the characterization of 21 secondary metabolites belonging to methoxylated flavonoids, phenolic acids, anthocyanins, and coumarins.</p> Conclusion <p><i>C. japonica</i> gel exhibits significant efficacy in wound healing in vivo and warrants further investigation for potential clinical applications.</p>