The aim of this study was to compare the effect of the Er,Cr:YSGG laser and different cavity disinfection agents on microleakage of an etch-and-rinse and a self-etch adhesive. Class V preparations were completed on the buccal and lingual surfaces of 30 extracted noncarious human molars. The occlusal margin was placed on enamel and the gingival margin on dentin. Preparations were randomly divided into five experimental groups (n=12); (1) 2% chlorhexidine gluconate (CHX), (2) propolis, (3) ozone, (4) Er,Cr:YSGG laser, and (5) control (no treatment). Each group was divided into two subgroups according to the adhesive system: etch-and-rinse (Adper Single Bond 2), and a self-etch adhesive (All-Bond SE). The preparations were bulk-filled with a resin composite (Arabesk). After storage in distilled water for 24 h the restored teeth were subjected to thermocycling (1,000 cycles; 5-55°C). All specimens were immersed in 0.5% basic fuchsin solution for 24 h and sectioned longitudinally through the centre of the restorations and examined under a stereomicroscope at ×25 magnification. The data were analysed using the Kruskal-Wallis and Mann-Whitney Utests. No difference was observed between the groups either on enamel or dentin when the etch-and-rinse adhesive was used (p>0.05). In the self-etch adhesive groups, a significant difference was found only between the laser group and the CHX group on enamel and between the propolis group and the control group on dentin (p<0.05). Comparing the etch-and-rinse and self-etch adhesives within each group, no differences were found on dentin (p>0.05). On enamel, a statistically significant difference was found only in the CHX group (p<0.05). There were no differences in microleakage with the laser and the different cavity disinfectant applications when used with etch-and-rinse adhesive. In the self-etch group there were differences in microleakage depending on the disinfection agent used. © Springer-Verlag London Ltd 2011.