Objective: The aim of this study was to evaluate the microleakage of composite resin restorations using two different dentine adhesive systems prepared with a diamond instrument and different parameters of Er:YAG laser irradiation. Background Data: Information on this topic with regard to preparing class V cavities with different parameters of Er: YAG laser irradiation and adhesive systems is scarce. Materials and Methods: Two hundred class V cavities were assigned to ten groups (n = 20 each): group 1: Er: YAG laser (5 Hz, 600 mJ) + phosphoric acid (PA) + Adper Single Bond 2 (ASB2); group 2: Er: YAG laser (10 Hz, 300 mJ) + PA + ASB2; group 3: Er: YAG laser (15 Hz, 200 mJ) + PA + ASB2; group 4: Er: YAG laser (20 Hz, 150 mJ) + PA + ASB2; group 5: diamond instrument + PA + ASB2; group 6: Er: YAG laser (5 Hz, 600 mJ) + Adper Prompt L-Pop (APLP); group 7: Er: YAG laser (10 Hz, 300 mJ) + APLP; group 8: Er: YAG laser (15 Hz, 200 mJ) + APLP; group 9: Er: YAG laser (20 Hz, 150 mJ) + APLP; and group 10: diamond instrument + APLP. Cavities were restored with a nanofill composite (Filtek Supreme XT Body). After thermocycling, the specimens were stained with 0.5% aqueous basic fuchsin dye and sectioned bucco-lingually. Dye penetration was then scored. The data were analyzed using the Kruskal-Wallis and Mann-Whitney U tests with Bonferroni correction. The Wilcoxon signed ranks test was used to compare occlusal and gingival scores. Results: Leakage was seen in all groups at both the occlusal and gingival margins. The Kruskal-Wallis test showed statistically significant differences among the 10 groups (p < 0.001). The gingival margins had more microleakage than the occlusal margins (p < 0.001). Pairwise analysis by the Mann-Whitney U test showed that statistically significant differences (p < 0.05) in microleakage were found between groups 3 and 5 (3 > 5), 5 and 7 (7 > 5), and 7 and 8 (7 > 8) at the gingival margin, and between groups 3 and 6 (6 > 3), 3 and 7 (7 > 3), 4 and 6 (6 > 4), and 4 and 7 (7 > 4) at the occlusal margin. Conclusion: We concluded that for all groups, microleakage values were higher at the gingival margins. The use of the Er: YAG laser for cavity preparation with different parameters and different dentine adhesive systems influenced the marginal sealing of composite resin restorations.