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. Topical treatment can 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 of 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 CO 2 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 <1 ms). Results: Imaging revealed a three-layered nail structure, an AFL-induced porous ablation crater, and changes in autofluorescence. While intact fungal samples showed a 106% higher ATTO-647N signal intensity than healthy controls, microporation led to a significantly increased fluorophore permeation in all samples (p < 0.0001). AFL processing of nail tissue enhanced topical delivery of ATTO-647N in all layers, (average increase: healthy +108%, fungal +33%), most pronounced in the top nail layer (healthy +122%, fungal +68%). While proportionally deeper ablation craters correlated moderately with higher fluorescence intensities in healthy nail tissue, fungal samples showed no significant relationship. Conclusion: Fractional CO 2 laser microporation is a simple way of enhancing the passive delivery of topically applied ATTO-647N. Although the impaired nail