This study aimed to assess the in vitro efficacy of the lasers Er:YAG, Nd:YAG, and CO(2) operating in the low energy mode for caries prevention in pits and fissures. Forty-five caries-free enamel occlusal sections were randomly divided into three groups: G1 - Er:YAG (80 mJ/2 Hz); G2 - Nd:YAG Laser (1 W and 10 Hz); and G3 - CO(2) Laser (0.4 W and 20 Hz). After surface treatment, the samples were submitted to challenge with acid consisting of a 10-day immersion in demineralizing (6 h) and remineralizing solution (18 h). Next, enamel demineralization was quantitatively evaluated by subsurface microhardness test and polarized-light microscopy (PLM, mm(2)) and qualitatively assessed by scanning electron microscopy. The Wilcoxon test was used for comparison of each group with its own control. ANOVA (α = 5%) was employed for comparison among groups, and Fisher's LSD multiple comparison test was applied, to check the difference in means. Concerning the microhardness analyses, statistical difference between control, and experimental areas was only detected for the CO(2) group. Experimental values were higher than the controls. As for PLM analyses, smaller demineralized areas were measured for G2 (Nd:YAG) and G3 (CO(2)) compared with the control areas. In conclusion, the present findings suggest that the CO(2) laser should be selected in order to increase the enamel resistance to acid in pits and fissures.