The aim of this study was to compare the effects of the Er,Cr:YSGG laser using chemical vapour deposition (CVD) bur cavity preparation with conventional preparation methods including a diamond bur and a carbide bur on the microleakage with two different adhesive systems. A total of 40 extracted human premolars were randomly assigned to four experimental groups according to the cavity preparation technique: group I diamond bur (Diatech); group II carbide bur (Diatech); group III Er,Cr:YSGG laser (Biolase Millennium II); and group IV CVD bur (CVDentUS). Using the different preparation techniques, Class V standardized preparations were performed on the buccal and lingual surfaces with gingival margins on the dentin and occlusal margins on the enamel. Each preparation group was randomly assigned to two subgroups (five teeth, ten preparations) according to the type of adhesive: an etch-and-rinse adhesive (Adper Single Bond), and a single-step self-etch adhesive (AdheSE One). All preparations were restored with a nanohybrid composite resin in a single increment. Following thermocycling (×500; 5-55°C), the teeth were immersed in basic fuchsin and sectioned in the orovestibular direction. Dye penetration was evaluated under a light microscope by two blinded examiners. Data were statistically analysed with the Kruskal-Wallis and Wilcoxon tests (p<0.05). There were no statistically significant differences between the preparation techniques with either of the two adhesive systems (p>0.05). Comparing the enamel and dentin leakage scores within each group, no statistically significant differences were found (p>0.05). The Er,Cr:YSGG laser cavity preparation did not differ from preparation with CVD, diamond or carbide bur in terms of microleakage with the different adhesive systems.