This study aimed to evaluate the microleakage of a new universal adhesive used in self-etch (Se) and etch-and-rinse (Er) mode compared with conventional adhesive systems. Fifty standard Class V caries lesions were prepared on the buccal and lingual surfaces of premolar teeth and randomly divided into five groups (n = 10): Group 1 (control), Adper Single Bond 2 (SB); Group 2, Single Bond Universal applied as Er (SBUEr); Group 3, Single Bond Universal applied as Se (SBUSe); Group 4 (control), Clearfil SE (CSE); and Group 5, Xeno V (XE). On both enamel and dentin, XE showed the highest microleakage scores, and SBU showed similar leakage with the control group.