According to the results of the clinical trials, laser therapy is effective for the treatment of onychomycosis, but the in vitro findings are inconsistent among studies. This study aimed to explore the experimental conditions of laser for the inhibition of Trichophyton rubrum growth in vitro. A 1064-nm neodymium-doped yttrium aluminum garnet (Nd:YAG) laser was used to irradiate colonies using a small (6-mm diameter) or large (13-mm diameter) area, and using 300, 408, or 600 J/cm2. The surface temperature of the colony was measured after irradiation and every 2 min. The growth area was calculated until the 7th or 10th day of incubation daily.For the small area group, at 300 J/cm2, the immediate surface temperature was 25.2 ± 0.2°C, but without effect on growth (P = 0.516). At 408 J/cm2, the immediate surface temperature was 32.0 ± 0.4°C; growth was inhibited for 7 days (P < 0.001). At 600 J/cm2, the immediate surface temperature was 38.1 ± 0.4°C; the growth was completely stopped for at least 10 days (P < 0.001). For the large area group, the temperature patterns were similar to those of the small area group, but the highest temperature was lower than in the small area groups, and no growth inhibition effect was observed (all P > 0.05).When the irradiation area is small, a 1064-nm Nd:YAG laser at 408 or 600 J/cm2 can be effective in suppressing T. rubrum growth in vitro.© 2022. The Author(s).