The sensation of pain in wounds is attributed to the secretion of Substance P (SP), an 11-amino acid neuropeptide that functions as both a neurotransmitter and a neuromodulator. SP is produced by the central nervous system, peripheral nervous system, and immune cells. Research is also underway to study suppression of SP activity as a potential treatment for pain. Medical plasma can mitigate discomfort during wound healing. The ability of plasma therapy to expedite healing and diminish infections may alleviate discomfort during wound healing. The decrease in pain can be quantified by measuring the SP levels during medical plasma treatment. The objective of this study was to investigate the efficacy of medical plasma therapy in reducing pain in methicillin-resistant Staphylococcus aureus (MRSA)-infected wounds by analyzing SP levels and fibroblast, neutrophil, and platelet levels as supporting data for SP. In this investigation, a prototype of a medical plasma technology of the atmospheric pressure jet type was categorized into four treatment groups: control (K), group with plasma treatment at a distance of 5 mm [control plasma (CP)], group with plasma treatment at a distance of 20 mm [noncontact plasma (NCP)], and group with plasma treatment at a distance of 5 and 20 mm (CP-NCP). The study revealed that SP levels decreased in the treatment groups administered medical plasma (CP, NCP, and CP-NCP) compared to the control group during the treatment period (14 days), and SP levels significantly decreased on days 11 and 14. The lowest levels recorded on D14 were 517.755, 684.45, and 790.19 pg/mL, followed by 985.685, 1120.24, and 1205.905 pg/mL on day 11. A decrease in SP levels indicates loss of pain in the wound. Administration of medical plasma to MRSA-infected wounds had a positive effect on pain relief in mice, which also helped in faster wound healing.