This study aimed to evaluate the microleakage of a universal adhesive's different application modes incorporated with Er,Cr:YSGG laser on Class V resin composite restorations. Sound human molar teeth (n= 30) were used for microleakage evaluations. Specimens with 60 standardized Class V cavities were divided into five groups according to the adhesive modes of universal adhesive, Adhese Universal (n= 12). Group 1-etch-and-rinse mode with phosphoric acid; Group 2-etch-and-rinse mode with Er,Cr:YSGG laser; Group 3-selective-etch mode with phosphoric acid; Group 4-selective-etch mode with Er,Cr:YSGG laser; Group 5-self-etch. After restorations were performed with a resin composite, Tetric N-Ceram, the specimens were polished and subjected to thermocycling (10,000X). Following immersion in 0.5% basic fuschin for a day, the teeth were sectioned and the degree of microleakage was determined along the tooth-resin composite interface using a light microscopy(40X). Five specimens from each group were examined by scanning electron microscopy. The Kruskal-Wallis, Siegel Castello, and Wilcoxon tests were used for statistical analyses (alpha= .05). At the enamel margins, significant differences were obtained among the groups (p< .05). Significantly higher microleakage scores were detected in Group 5 in comparison with Groups 1, 2, and 3. There were no significant differences between different adhesive strategies at the dentin margins (p> .05). While analyzing enamel and dentin microleakage scores, no statistically significant differences were observed in Groups 4 and 5 (p> .05). The laser application time and the adhesive modes of universal adhesives could affect the microleakage at the enamel margins. Different adhesive modes of universal adhesives combined with laser etching had no influence on the microleakage scores of dentin margins.