Objective: Despite promising results of Er:YAG laser in periodontal debridement, to date there is no consensus about the ideal settings for clinical use. This experimental clinical trial aimed to determine the effects of debridement using Er:YAG laser and to compare with ultrasonic treatment. Materials and methods: Sixty-four teeth were divided into two in vivo and in vitro subgroups. Each tooth received ultrasonic treatment on one side and Er:YAG laser debridement at either 60, 100, 160 or 250mJpulse(-1) and at 10Hz on the other side on a random basis. All samples were morphologically analyzed afterwards under scanning electron microscope for surface changes and dentinal tubules exposure. Treatment duration (d) was also recorded. Results: Laser debridement produced an irregular, rough and flaky surface free of carbonization or meltdown while ultrasound produced a relatively smoother surface. The number of exposed dentinal tubules (n) followed an energy-dependent trend. The number of exposed tubules among the in vivo laser groups was n60mJ=n100mJd100mJ>dUltrasound=d160mJ>d250mJ (P0.046), while for the in vitro groups it was d60mJ>d100mJ=dUltrasound=d160mJ>d250mJ (P0.046). Conclusions: Due to excessive treatment duration and surface damage, Er:YAG laser debridement at 60 and 250mJpulse(-1), respectively, is not appropriate for clinical use. Although laser debridement at 100 and 160mJpulse(-1) seems more suitable for clinical application, compared to ultrasound the former is more time-consuming and the latter is more aggressive. Using a feedback device or lower pulse energies are recommended when using laser in closed field.