Laser treatment has emerged as a novel treatment modality for onychomycosis. We sought to determine thermal response and optical effects of a submillisecond neodymium:yttrium-aluminum-garnet (Nd:YAG) 1064-nm laser on common fungal nail pathogens, and the clinical efficacy and safety of the Nd:YAG 1064-nm laser on onychomycotic toenails. A 4-part in vitro and in vivo study was conducted using a Nd:YAG 1064-nm laser. The first portion evaluated 3 different nail pathogens in suspension at 7 heat and time exposures. The second and third parts of the study irradiated pure fungal colonies. The final portion involved an in vivo treatment of toenails over 5 treatment sessions. A fungicidal effect for Trichophyton rubrum was seen at 50°C after 15 minutes, and for Epidermophyton floccosum at 50°C after 10 minutes. Limited growth of Scytalidium was seen at 55°C after 5 minutes. No inhibition was observed after laser treatment of fungal colonies or suspensions. In vivo treatment of toenails showed no improvement in Onychomycosis Severity Index score. The Nd:YAG 1064-nm laser was the only laser tested. Laser treatment of onychomycosis was not related to thermal damage or direct laser effects. In vivo treatment did not result in onychomycosis cure.