Objective: The aim of this study was to evaluate the microleakage of different nano-restorative materials in Class V cavities prepared by Er:YAG laser and bur preparation. Materials and Methods: Class V cavities were prepared on the buccal and lingual surfaces of 72 premolars by Er:YAG laser or bur. The occlusal margins were in enamel and the cervical margins were in cementum. Teeth were randomly assigned to six groups of 12 teeth (n = 24 cavities) each as follows: Group 1, Er:YAG laser preparation (E)+Ketac N100 (K); Group 2, bur preparation (B)+K; Group 3, E+Adper Prompt L-Pop (A)+Filtek Supreme XT Flow (FSF); Group 4, B+A+FSF; Group 5, E+A+Filtek Supreme XT (FS); Group 6, B+A+FS. All teeth were thermocycled 500 times. Ten teeth from each group were chosen for the microleakage investigation and two teeth for the scanning electron microscope evaluation. Teeth prepared for the microleakage test were immersed in 0.5% basic fuchsin dye for 24 h. Afterwards, the teeth were sectioned and observed under a stereomicroscope for dye penetration. Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (p < 0.05). Results: There were significant differences between occlusal and cervical regions for all groups (p < 0.05) except for Group 1. Bur-prepared cavities showed less microleakage in all groups for enamel (p < 0.05); however, in cementum there were no significant differences between the bur- and laser-prepared cavities in nano-glass ionomer and flowable composite groups (p > 0.05). Conclusion: It may be concluded that the cavities prepared by Er:YAG laser showed higher degree of microleakage than those conventionally prepared by bur, regardless of the restorative material at enamel margins.