This in vitro study evaluated the compositional, crystalline, and morphological effects promoted by Nd:YAG laser on root dentin, and verified the effects of laser and topical acidulated phosphate fluoride application (APF-gel) on dentin erosion. 180 bovine dentin slabs were randomized into 4 groups (n = 45): G1–untreated, G2–APF-gel (1.23% F−, 4 min), G3–Nd:YAG (1064 nm, 84.9 J/cm2, 10 Hz), and G4–APF-gel application followed by Nd:YAG laser irradiation. The compositional, crystalline, and morphological effects promoted by treatments were investigated on five samples of each experimental group. The other samples were submitted to a 5-day, 10-day, or 15-day erosive and abrasive demineralization and remineralization cycling in order to create erosion lesions. The area and depth of lesions, as well as the optical attenuation coefficient, were assessed, and all data were statistically analysed (p < 0.05). Nd:YAG laser promoted the reduction of carbonate, the formation of tetracalcium phosphate, as well as the melting and recrystallization of the dentin surface. Laser significantly decreased the area and depth of erosion lesions and altered the optical attenuation coefficient when compared to untreated and APF-gel groups, but the association of APF-gel and laser did not promote an additional effect. Nd:YAG laser irradiation can be a promissory treatment to prevent dentin erosion and the abrasion process.