The present study evaluates the degree of apical leakage in vitro after root canal preparation using Er:YAG laser irradiation followed by obturation. Twenty-four single-rooted teeth were divided into 2 groups of 12. One group served as a control and these root canals were conventionally prepared up to a #50K file. The other group was prepared by Er:YAG laser irradiation at parameters of 2 Hz and 170 to 230 mJ/pulse. After obturation the teeth were immersed in a vacuum flask containing 0.6% rhodamine for 48 h, longitudinally bisected, and observed by stereoscopy and scanning electron microscopy. The degree of apical leakage from an apical stop was measured and statistical analysis was performed. The degree of apical leakage from the teeth prepared by laser was not significantly less than that from control teeth (p > 0.01). Morphological findings showed that contact between the root canal walls and obturated materials was hermetic in both groups, but canal walls prepared by laser were rough and irregular. These results show that root canal preparation by laser does not affect apical leakage after obturation compared with leakage in canals prepared using the conventional method.