Onychomycosis is one of the most common nail disorders. Its current treatment is not satisfactorily effective and often causes adverse side effects. This study aims to determine the optimal conditions for non-thermal plasma (NTP) inactivation of the most common dermatophytes in vitro and to apply it in patient`s therapy. The in vitro exposure to NTP produced by negative DC corona discharge caused full inactivation of Trichophyton spp. if applied during the early growth phases. This effect decreased to negligible inactivation with the exposure applied six days after inoculation. In a group of 40 patients with onychomycosis, NTP therapy was combined with nail plate abrasion and refreshment (NPAR) or treatment with antimycotics. The cohort included 17 patients treated with NPAR combined with NTP, 11 patients treated with antimycotics and NTP, and 12 patients treated with NPAR alone. The combination of NPAR and NTP resulted in clinical cure in more than 70% of patients. The synergistic effect of NPAR and NTP caused 85.7% improvement of mycological cure confirmed by negative microscopy and culture of the affected nail plate. We conclude that NTP can significantly improve the treatment of onychomycosis.