The purpose of this study was to evaluate the efficiency of the Er:YAG laser and diamond bur cavity preparation on the marginal microleakage of Class V cavities. Group 1: bur preparation (bp)+Vertise Flow (VF); Group 2: laser preparation (lp)+VF; Group 3: bp+Adper Easy One (AEO)+Filtek Ultimate Flowable Composite (FUFC); Group 4: lp+AEO+FUFC; Group 5: bp+Clearfil S3 Bond (CSB)+Clearfil Majesty Flow (CMF); Group 6: lp+CSB+CMF. Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (p<.05). More microleakage was observed in cervical regions compared to occlusal regions in all groups (p<.05). No significant difference was observed among all groups in terms of occlusal and cervical surfaces, respectively (p>.05). The use of the Er:YAG laser for cavity preparation with different adhesive systems and flowable composites did not influence microleakage.