This study aimed to assess how the wear that brushing promotes affects CO 2 laser-irradiated enamel microhardness after cariogenic challenge in vitro. Forty fragments measuring 4×4 mm were randomly assigned to four groups according to the enamel surface treatment: G1—control, G2—CO 2 -laser irradiation, G3—brushing, and G4—CO 2 laser irradiation + brushing. A laser device emitting at 10.6 μm was used (power=0.5 W , energy per pulse=0.05 mJ , and frequency=10 kHz ). Specimens belonging to groups G3 and G4 were brushed (80,000 strokes) with a brushing simulator using toothpaste. Next, the samples were challenged with acid: the specimens were immersed in demineralizing and remineralizing solutions for 8 days. The acid resistance of enamel was evaluated by cross-sectional microhardness tests. The area under the curve (KHN×μm ) was calculated. Analysis of variance (ANOVA) one-away and Fisher’s test were performed for the statistical analysis (p<0.05 ). Group G2 specimens (31,185±4706 ) were statistically different from specimens belonging to groups G1 (26,723±2446 ), G3 (28,194±1376 ), and G4 (28,207±2234 ), which were statistically similar. The brushing time used in the present study probably wore the CO 2 -lased enamel, so demineralization could not be prevented in the brushed group.