This in vitro study compared the microleakage of Class V resin composite restorations placed in cavities prepared with a high-speed dental bur, air-abrasion or Er:YAG laser. Twenty sound extracted human third molars were selected and randomly assigned to four equal Groups (n=10): Group I, cavities were cut by dental drill at highspeed; Group II, aluminum oxide air-abrasion was used for cavity preparation, and in Groups III and IV, cavities were prepared by Er:YAG laser. Following cavity preparation, Groups I and II were acid-etched, Group III was treated only by Er:YAG laser and Group IV was conditioned by Er:YAG laser followed by acid-etching. Cavities were restored (Single Bond + Z-100) and the teeth stored for seven days in distilled water. Then, the restorations were polished and the specimens thermocycled, immersed in a 0.2% Rhodamine solution, sectioned and analyzed for leakage at the occlusal (enamel) and cervical (dentin/cementum) interfaces using an optical microscope connected to a video camera. The images were digitized and software was utilized for microleakage assessment. Upon analyzing the results, statistically significant differences (p <0.01) between the occlusal and cervical regions for all groups was observed, and, as a rule, there was better marginal sealing at the enamel margins. The highest degree of infiltration was observed for cavities prepared and treated exclusively by Er:YAG (Group HI). The other experimental groups showed statistical similarities in the amount of marginal leakage at the enamel margins. However, at the cervical margins, there was a significant difference (p <0.05) between Group I and the remaining groups. None of the techniques completely eliminated marginal microleakage at the dentin/cementum margins.