This study aimed to evaluate the effects of repeated CO2 laser applications on the inhibition of enamel demineralization. Sixty-five human dental enamel slabs were randomly assigned to the following groups (n = 13): control (C), one application of the CO2 laser (L1), two applications of the CO2 laser (L2), three applications of the CO2 laser (L3), and four applications of the CO2 laser (L4). Enamel slabs were irradiated by a 10.6-μm CO2 laser operating at 5 J/cm(2). The slabs were subjected to a pH-cycling regimen and then analyzed by FT-Raman spectroscopy, energy-dispersive X-ray fluorescence spectrometry (EDXRF), cross-sectional micro-hardness, and scanning electron microscopy (SEM). Statistical analysis was performed using ANOVA and Tukey tests (p < 0.05). FT-Raman spectroscopy showed a reduced carbonate content for L1, L3, and L4 groups when compared to C (p < 0.05). The EDXRF data showed no statistical differences between the control and irradiated groups for calcium and phosphorus components (p > 0.05). Cross-sectional micro-hardness data showed a statistically significant difference between the control and all irradiated groups (p < 0.05), but no difference was found among the irradiated groups (p > 0.05) up to 30-μm depth. A tendency of lower demineralization occurred in deeper depths for L3 and L4 groups. The SEM results showed that with repeated applications of the CO2 laser, a progressive melting and recrystallization of the enamel surface occurred. Repeated irradiations of dental enamel may enhance the inhibition of enamel demineralization.