The erbium:yttrium–aluminum–garnet (Er:YAG) laser is now increasingly used in periodontal therapy. The purpose of this study was to investigate the effect of Er:YAG laser irradiation on the morphology of periodontopathic bacteria and to compare the bacterial elimination effect of the laser and the ultrasonic scaler on diseased root surfaces in vitro. Colonies of Porphyromonas gingivalis were exposed to a single-pulse Er:YAG laser at 40 mJ and were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Also, 20 pairs of periodontally diseased root surfaces with subgingival calculi of freshly extracted teeth were treated by Er:YAG laser scaling at 40 mJ/pulse (14.2 J/cm² per pulse) and 10 Hz with water spray or ultrasonic scaling, or were not treated. The efficiency of each treatment was determined as the area treated per second, and the treated surfaces were examined by SEM. The material scraped from the treated root surfaces was cultured in aerobic and anaerobic conditions, and the numbers of colony forming units (CFUs) were compared. SEM and TEM showed that the Er:YAG laser had easily ablated the bacterial colony, leaving an ablation spot with a crater and the surrounding affected area showing melted branch-like structures. The laser irradiation was as equally effective and efficient as the ultrasonic scaler in performing root surface debridement. The CFUs after laser treatment were significantly fewer than those after ultrasonic scaling in aerobic and anaerobic culture conditions. Er:YAG laser ablates periodontopathic bacteria with thermal vaporization, and its bacterial elimination effect on the diseased root surfaces appears to be superior to that of the ultrasonic scaler.