Background: Nonthermal atmospheric plasma (NTAP) has been studied as a new treatment option for various medical conditions. Onychomycosis is a common nail disorder caused by a fungus that is difficult to treat and often recurs. One reason for treatment difficulty is the growing resistance to current antifungal medications. Given the medical concerns related to antifungal drugs, there is a need for newly developed treatment options. Objective: This study sought to determine if NTAP was effective as an antifungal treatment for onychomycosis. To explore this innovative treatment option, we conducted in vitro and ex vivo experiments using microbial methods and microscopy. Methods: For the in vitro experiments, Trichophyton (T.) rubrum was inoculated into Sabouraud dextrose agar plates. For the ex vivo experiments, nail specimens diagnosed with onychomycosis resulting from T. rubrum were used. NTAP using argon gas was utilized in this study. Scanning electron and reflectance confocal microscopy were used to analyze the nail specimens. Results: The growth of T. rubrum colonies exposed to NTAP was markedly suppressed compared to untreated colonies in vitro. T. rubrum ultra-structures noticeably transformed from smooth to rough or ruptured after exposure. Conclusion: NTAP can inhibit the growth of T. rubrum and cause damage to fungal spores. This finding could potentially lead to the development of new and effective treatments for onychomycosis.