Objective: The aim of this study was to evaluate the influence of cavity preparation with different Er,Cr:YSGG laser handpieces on microleakage of different posterior composite restorations. Methods: Fifty-four extracted intact human premolars were randomly assigned to three groups according to cavity preparation method: Bur Group: high-speed diamond bur (Diatech), MD Group: Er,Cr:YSGG laser Waterlase MD handpiece (Biolase Millennium II), and Turbo Group: Er,Cr:YSGG laser Waterlase MD TURBO handpiece (Biolase Millennium II). One hundred eight Class II slot cavities were prepared on the mesial and distal proximal surfaces of each tooth, and the cavity preparation times required were determined. The groups were then subdivided according to the restorative systems used (n=12): a conventional methacrylate-based microhybrid composite (Filtek P60+Adper Single Bond 2/3M); a silorane-based resin composite (Filtek Silorane+Silorane System Adhesive/3M); and a nanohybrid methacrylate-based composite (Kalore+G-Bond/GC). The restorative systems were applied according to the manufacturers' recommendations. Following thermocycling (X5000; 5 degrees C-55 degrees C), the teeth were coated with nail varnish except the restoration margins, immersed in 0.5% basic fuchsin dye solution, and sectioned in a mesiodistal direction. Dye penetration was evaluated under a light microscope for occlusal and cervical margins. Data were analyzed with one-way ANOVA and chi-square tests (p<0.05). Results: The cavity preparation time (mean +/- SD) required for Bur, MD, and Turbo group was 31.25 +/- 3.82, 222.94 +/- 15.85, and 92.5 +/- 7.42sec, respectively, and the differences among the groups were statistically significant (p<0.05). Comparing the occlusal and cervical microleakage scores, no statistically significant differences were found among the groups and subgroups (p>0.05). Conclusions: Er;Cr:YSGG laser cavity preparation with the Turbo handpiece needed shorter time than the MD handpiece, although it needed longer time than the conventional diamond bur. The use of different handpieces of Er,Cr:YSGG laser did not differ from conventional preparation with diamond bur in terms of microleakage with the tested methacrylate- and silorane-based posterior composite restorative systems.