This study aimed to investigate the impact of ablative fractional laser (AFL) on hedgehog pathway gene expression in murine microscopic basal cell carcinomas (BCCs) and compare these results to the effect of topical treatment with vismodegib, an FDA-approved hedgehog inhibitor. In 25 mice, 1 cm² skin test sites (n = 44) containing microscopic BCCs were exposed to one of three interventions: a single CO₂ AFL treatment (1 pulse, 40 mJ/microbeam, wavelength 10.6 μm, 5% density, pulse rate 250 Hz, n = 12), eight topical vismodegib treatments (3.8 mg/mL, n = 8), or combination of AFL and vismodegib treatments (n = 9). Untreated controls were included for comparison (n = 15). After 4 days, skin samples were analyzed for hedgehog gene expression (Gli1, Gli2, and Ptch1) by qPCR and vismodegib concentrations by liquid chromatography mass spectrometry (data analyzed with two-tailed t-tests and linear regression). A single treatment with AFL monotherapy significantly reduced hedgehog gene expression compared to untreated controls (Gli1 72.4% reduction, p = 0.003; Gli2 55.2%, p = 0.010; Ptch1 70.9%, p < 0.001). Vismodegib treatment also reduced hedgehog gene expression (Gli1 91.6%; Gli2 83.3%; Ptch1 83.0%), significantly surpassing AFL monotherapy for two out of three genes (Gli1, p = 0.017; Gli2, p = 0.007; Ptch1, p = 0.15). AFL and vismodegib combination mirrored the effects of vismodegib monotherapy (Gli1, p = 0.424; Gli2, p = 0.289; Ptch1, p = 0.593), possibly due to comparable cutaneous vismodegib concentrations (mean ± SD, vismodegib monotherapy 850 ± 475 µmol/L; combination 1036 ± 824 µmol/L; p = 0.573). In conclusion, a single AFL treatment significantly reduced hedgehog gene expression in murine BCCs mimicking the effects of eight topical applications of vismodegib. Further studies are needed to assess whether AFL can be utilized for BCC treatment, either as monotherapy or in combination with other drugs.