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Abstract Background This in-vitro study aimed to investigate the effect of two different antibacterial disinfectants on the microleakage performance of newly developed bulk-fill composite, bonded to different tooth structures. Methods Class V cavities were prepared in 30 sound premolar teeth, with enamel occlusal margins (OM) and dentin cervical margins (CM). Two disinfectants, 2% chlorhexidine gluconate (CHX) and Listerine Miswak (ListM), were used. Teeth were divided into three groups (n = 10): G1, Control; G2, CHX; and G3, ListM. Disinfectants were applied to the cavity preparation walls after they were etched with 35% phosphoric acid. The Single Bond Universal adhesive system was then used, and teeth were restored with Filtek One Bulk Fill composite. Samples were examined, after thermocycling aging, by stereomicroscopy for the evaluation of marginal dye penetration. Results The highest mean microleakage score was reported in the CM of G1 (2.60 ± 1.174), which was significant compared with that of G2 only (p = 0.02). OM in G1 showed no microleakage, with no significant differences found among groups (χ2 = 1.39, p = 0.50). No significant differences were reported between G2 and G3 (p = 0.45 OM; p = 0.17 CM). Conclusions Cavity pretreatment with CHX is not significantly different to pretreatment with CHX. In contrast, CHX improved the cervical marginal seal as compare to the control group (G1).