Purpose Conventional oral antifungal therapies for onychomycosis (OM) often do not achieve complete cure and may be associated with adverse effects, medical interactions, and compliance issues restricting their use in a large group of patients. The topical treatment provides an alternative to bypass the systemic side effects but is limited by the physical barrier of the nail plate. Ablative fractional laser (AFL) treatment can be used to improve the penetration of topical drugs into the nail. This study visualized the effects of laser ablation on nail tissue and assessed their impact on the biodistribution of a fluorescent dye in healthy and fungal nail tissue. Methods For the qualitative assessment of CO2 AFL effects on healthy nail tissue, scanning electron microscopy (SEM), coherent anti-Stokes Raman scattering microscopy (CARS-M), and widefield fluorescence microscopy (WFM) were used. To quantitate the effect of laser-pretreatment on the delivery of a fluorescent dye, ATTO-647N, into healthy and fungal nail tissue, ablation depth, nail plate thickness, and ATTO-647N fluorescence intensity in three nail plate layers were measured using WFM. A total of 30 nail clippings (healthy n = 18, fungal n = 12) were collected. An aqueous ATTO-647N solution was directly applied to the dorsal surface of 24 nail samples (healthy n = 12, fungal n = 12) and incubated for 4 hours, of which half (healthy n = 6, fungal n = 6) had been pretreated with AFL (30 mJ/mb, 15% density, 300 Hz, pulse duration