(1) Background: Numerous studies have shown the beneficial role of fluoride in the primary prevention of dental caries. The aim of the present study was to put into evidence the change in the enamel structure immediately after the application of a fluoride varnish. (2) Methods: A xylitol−fluoride varnish was evaluated. The enamel specimens (n = 10) were analyzed by atomic force microscopy on enamel surface and treatment with fluoride varnish applied. The dimensional topographic analysis was performed by 2D and 3D analysis software. Statistical analysis was performed using SPSS Version 26.00 (IBM, Armonk, NY, USA). A one-sample statistics test was used to identify significant differences (p < 0.05). (3) Results: Surface roughness (Ra) measurements ranged from Ra = 0.039 μm (±0.048), to Ra = 0.049 μm (±0.031), respectively (p < 0.05), with an increase in the surface roughness passing from the intact enamel to the enamel exposed to fluoride varnish. When comparing Ra values of the nonfluorinated enamel and fluorinated enamel, significant differences (p < 0.05) were found, suggesting that the varnish had a protective effect on the enamel surface. (4) Conclusions: Xylitol−fluoride varnish, even in one single short-time application, is effective in reducing the surface roughness of enamel structure exposed to abrasion, thus increasing resistance to dental caries.