This study aimed to assess how the wear that brushing promotes affects CO₂ 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-CO₂-laser irradiation, G3-brushing, and G4-CO₂ 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 CO₂-lased enamel, so demineralization could not be prevented in the brushed group.