Objective: To evaluate the effect of chlorhexidine and ethanol on microleakage of composite resin restoration to dentine. Methods: Class II cavities with dentinal margin were prepared on 96 premolar teeth. All specimens were acid-etched, rinsed and dried. Then the samples were randomly divided into four groups according to pre-treatment of the dentine: no treatment (control group); treatment with 100% ethanol for 60 s (group 2); treatment with 2% chlorhexidine for 60 s (group 3); 100% ethanol for 60 s and then 2% chlorhexidine for 60 s (group 4). After dentine treatment, each group was bonded and restored with a universal micro hybrid composite resin, according to the manufacturers' recommendation. Microleakage was evaluated by dye extraction method in two subgroups, immediately (24 h) and after 6 months in storage. Scan electronic microscope analyses for two samples of each group were also conducted. Data were analysed by two-way analysis of variance and Tukey test. Results: The lowest and the highest amount of microleakage were observed in the ethanol group and in the control group, respectively. There were significant differences in microleakage among the groups (P = 0.003) and between measurement times (P = 0.001). For each storage time, the control group showed significant differences from the other groups and there were no differences between the other groups. Conclusion: Ethanol-wet bonding and chlorhexidine application may have potential benefits in lowering the occurrence of microleakage in the long term. Keywords: chlorhexidine, ethanol, microleakage, surface treatment