Diabetes and its complications, such as delayed wound healing, are increasing at an alarming rate in India, putting an enormous strain on the country’s limited healthcare resources. Hence, the present study proposes to screen/identify the possible mechanisms and to study the effect of the flavonoid-enriched fraction of Selaginella bryopteris extract against human keratinocyte cell lines (HaCaT) and streptozocin (STZ)-induced diabetic wounds in a male Wistar rat model. Chemical profiling was performed by an MTT assay. The obtained GC–MS analysis results showed the presence of amentoflavone, gallic acid, imidazole, palmitic acid, catechine, L-fucitol, lupeol, and myo-inositol as the major bioactive phytoconstituents. S. bryopteris induces the generation of ROS, the condensation of chromatin in the nucleus, and changes in the membrane potential of mitochondria in HaCaT cell lines. An S. bryopteris-dependent induction of apoptosis-mediated cell death in HaCaT cell lines was confirmed by an AO/PI analysis. Mitochondrial depolarization was reflected in JC-1 staining of cells. The wound size was reduced and epithelialization was enhanced. Keratinocyte migration decreased interleukins, TNF-α, IL-2, and IL-6 and the expression of pro-apoptotic (p53, caspase-3, caspase-9, and Bax) and anti-apoptotic (Bcl-2) genes in a dose-dependent manner. Keratinocyte migration increased antioxidant enzyme levels (CAT, SOD, MDA, and GSH). Wound healing is facilitated through the mitochondria-mediated apoptosis pathway, revealing a new area of diabetic wound therapy.