Recently, the Er:YAG and CO(2) lasers have been applied in periodontal therapy. However, the characteristics of laser-irradiated root cementum have not been fully analyzed. The aim of this study was to precisely analyze the alterations of root cementum treated with the Er:YAG and the CO(2) lasers, using non-decalcified thin histological sections. Eleven cementum plates were prepared from extracted human teeth. Pulsed Er:YAG laser contact irradiation was performed in a line at 40 mJ/pulse (14.2 J/cm(2)/pulse) and 25 Hz (1.0 W) under water spray. Continuous CO(2) laser irradiation was performed in non-contact mode at 1.0 W, and ultrasonic instrumentation was performed as a control. The treated samples were subjected to stereomicroscopy, scanning electron microscopy (SEM), light microscopy and SEM energy dispersive X-ray spectroscopy (SEM-EDS). The Er:YAG laser-treated cementum showed minimal alteration with a whitish, slightly ablated surface, whereas CO(2) laser treatment resulted in distinct carbonization. SEM analysis revealed characteristic micro-irregularities of the Er:YAG-lased surface and the melted, resolidified appearance surrounded by major and microcracks of the CO(2)-lased surface. Histological analysis revealed minimal thermal alteration and structural degradation of the Er:YAG laser-irradiated cementum with an affected layer of approximately 20-μm thickness, which partially consisted of two distinct affected layers. The CO(2)-lased cementum revealed multiple affected layers showing different structures/staining with approximately 140 μm thickness. Er:YAG laser irradiation used with water cooling resulted in minimal cementum ablation and thermal changes with a characteristic microstructure of the superficial layer. In contrast, CO(2) laser irradiation produced severely affected distinct multiple layers accompanied by melting and carbonization.