Cold atmospheric direct plasma (CADP), an ionized gas at ambient temperature, represents a promising approach to enhancing tissue regeneration. A laboratory-based study was conducted to investigate the effects of medical CADP on the reparative potential of full-thickness acute skin wounds in murine models. For the in vivo investigations, two full-thickness dermal injuries were induced in each murine subject, each with a diameter of approximately 8 mm (n = 20). We employed a floating electrode within a CADP system that generates atmospheric pressure air plasma, characterized by a plasma temperature ranging from 36 to 38°C. The dermal wounds received three plasma treatments, administered twice daily for irradiation durations of 5, 15, and 25 seconds. These wounds were subsequently evaluated at intervals of 2, 4, 6, 8, and 11 days post-wounding through histological examination and gene expression analysis. On the eleventh day, the wound healing rates were recorded at 34.80% for the control group, while the plasma-treated groups achieved rates of 56.62%, 84.97%, and 97.82%, respectively. Histological examination revealed that plasma-treatment promotes the development of epidermal keratin and granular strata, along with the formation of hair follicles and sebaceous glands. Gene expression analysis indicates increased levels of growth factors and a decrease in white blood cell counts. CADP therapeutic intervention has significantly enhanced the healing efficacy of acute dermatological lesions without any noticeable adverse effects or the simultaneous activation of pro-inflammatory signaling pathways. These findings underscore the benefits of employing plasma applications for wound management in clinical settings.