Investigations of laser- or light-assisted antibacterial and antifungal treatments have been introduced. In the present study, we investigated the antifungal activities of 1444-nm Nd:YAG lasers against onychomycosis by microbiologic analysis and scanning electron microscopy. Scraped toenails from 20 participants with mycologically confirmed onychomycosis were prepared on polystyrene weighing dishes and treated with a 1444-nm Nd:YAG laser. The samples were analyzed for the presence of colony-forming units (CFUs) and scanning electron microscopy was performed using an toenail treated with the 1444-nm Nd:YAG laser. The mean reduction rate achieved by treatment with a total energy of 300 J was 75.9% (range: 33.3-100), and by treatment with 450 J was 85.5% (range: 66.7-100). However, the difference in CFU reduction rates between the laser settings of 300 J and 450 J was not significant. Analysis by scanning electron microscope revealed numerous disintegrated spores on the lower portions of the nail plate treated with the 1444-nm laser, while the upper portion of the nail plate presented only a few small and greatly disintegrated fungal spores. Our results suggest that a Nd:YAG laser with a wavelength of 1444 nm has antifungal effects on onychomycosis. However, further investigations should be performed to determine the long-term clinical and microbiologic effects of this treatment.