The aim of this study was to analyze the effect of CO2 laser (10.6 μm) irradiation with 5-μs pulse width in prevention of enamel erosion due to citric acid exposure in vitro. One hundred forty-four bovine enamel samples were cut into 5 × 5 × 2-mm-size slabs and polished to obtain plane surfaces. Enamel surfaces were covered with acid-resistant varnish, except for a central area of 2.5 mm in diameter. The samples were divided into four groups (n = 12/group/day): C—control, no treatment; L—CO2 laser irradiation (0.3 J/cm2, 5 μs, 226 Hz); F—topical fluoride treatment, 1.25%F− (AmF/NaF) for 3 min; and FL—fluoride treatment + CO2 laser. For erosive demineralization, samples were immersed in 40 ml of citric acid (0.05 M, pH 2.3) for 20 min two times per day during 5 days. After 1, 3, and 5 days, surface loss was measured by digital profilometer. According to the repeated measure ANOVA and post hoc comparisons, all the treatments showed statistically significant reduction of enamel loss as compared to control group, in all investigated times (p < 0.0001): L (52%,31%,37%); F (28%,24%,29%); FL (73%,55%,57%). Both CO2 laser irradiation alone (L) and the combined laser–fluoride treatment (FL) caused less enamel loss than the fluoride group (F) in all days (p < 0.0001 for L in all times; and p < 0.0001, p = 0.0220 and p = 0.0051 for F, respectively, at days 1, 3, and 5). Under the conditions of this study, CO2 laser irradiation (0.3 J/cm2, 5 μs, 226 Hz) could effectively reduce enamel surface loss due to citric acid exposure, in vitro. This effect was still observed after 5 days of repeated acid exposures.