Topical therapy of nail psoriasis using methotrexate has not been realized due to the high molecular weight and low permeability of the compound. In this study, we used a 2940 nm fractional ablative laser to disrupt the nail barrier to enhance the in vitro transungual delivery of methotrexate. Bovine hoof membrane—an in vitro model of the human nail—was treated by the laser at different energy levels and pore densities. A successful microporation was characterized by mechanical properties, scanning electron microscopy, Fourier transform infrared spectrophotometer, dye binding, histology, pore uniformity, confocal laser microscopy, nail integrity measurement, and permeation studies. No significant difference in the pore dimension was found in different treatment groups (p > 0.05). Increases in pore depth corresponded with increases in the laser energy. Laser ablation was found to affect the mechanical properties of the hoof membrane. In in vitro permeation studies, laser ablation resulted in a significant increase in the drug cumulative delivery, flux, and permeability coefficient as compared to the untreated group (n = 3, p < 0.05). A change in the laser energy and pore density was found to alter the drug permeability. Thus, transungual methotrexate delivery was enhanced by the fractional laser ablation.