Onychomycosis is caused by dermatophyte infection of the nail. Though laser energy has been shown to eliminate dermatophytes in vitro, direct laser elimination of onychomycosis is not successful due to difficulties in selectively delivering laser energy to the deeper levels of the nail plate without collateral damage. Femtosecond (fsec) infrared titanium sapphire lasers circumvent this problem by the nonlinear interactions of these lasers with biological media. This quality, combined with the deeply penetrating nature of the near-infrared radiation, allows elimination of deeply seeded nail dermatopytes without associated collateral damage. Nail cuttings obtained from patients with onychomycosis caused by Trichophyton rubrum underwent fsec laser irradiation using increasing laser intensities with the focus scanned throughout the whole thickness of the nail specimen. The efficacy of the laser treatment was evaluated by subculture. Scanning electron microscopy was used to determine fsec laser-induced collateral damage. We found that a fsec laser fluence of 7 × 1031 photons m−2 s−1 or above successfully inhibited the growth of the fungus in all samples examined, whereas laser intensities above 1.7 × 1032 photons m−2 s−1 affected the structure of the nail plate. Our findings suggest that T. rubrum-mediated onychomycosis may be treated by fsec laser technology.