Plant Callus are a valuable source of pluripotent stem cells and bioactive phytochemicals. Meanwhile, the Hypericum perforatum callus extract (HPCE) is particularly rich in compounds such as hyperforin, hypericin, quercetin, and other phenolic and flavonoid derivatives. These phytochemicals exhibit strong antibacterial, antioxidant, anti-inflammatory, and anti-fibrotic properties, making them promising for wound healing. One of the most critical challenges following wound healing is the formation of fibrosis, which can compromise the complex structural integrity of skin. To address this issue, a poly(vinyl alcohol)/chitosan/alginate (PCA) wound dressing loaded with HPCE is developed. This hydrogel dressing features a porous structure with suitable mechanical properties and a high swelling capacity, potentially enhancing its effectiveness in promoting tissue regeneration and wound healing. In vitro studies have confirmed its biocompatibility, cell proliferation, and cell adhesion properties. Additionally, the dressing has demonstrated the ability to inhibit the proliferation of certain antibiotic-resistant bacteria. The in vivo studies revealed the anti-inflammatory properties, promotion of angiogenesis, facilitation of re-epithelialization, and stimulation of collagen deposition of the dressing under investigation. Moreover, the immunohistochemistry analysis of the two key markers, p16 and p53, has shown that the application of the dressing helps prevent fibrosis after wound healing.