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Objective To compare the disinfection effect of 3% (v/v) hydrogen peroxide and 500 mg/L chlorine-containing disinfectants in the independent waterway of a periodontal ultrasonic scaler to provide a reference for clinical waterway disinfection management in stomatology departments. Methods The 18 ultrasonic scalers were randomly divided into 3 groups of 6 units: the control group, experimental group 1 (3% hydrogen peroxide disinfectant group), and experimental group 2 (500 mg/L chlorine-containing disinfectant group). The replaceable parts of the independent waterway pipes of the 3 groups of ultrasonic scalers were replaced, and the water supply was supplied with sterile distilled water (DW). In the control group, special treatment was not applied to the nonreplaceable pipe part. In experimental group 1, the 3% hydrogen peroxide was used to disinfect nonreplaceable pipelines. In experimental group 2, the nonreplaceable part was disinfected with the 500 mg/L chlorine-containing disinfectant. The water sample was taken from the outlet of the scaler working part in the three groups for monitoring before disinfection, immediately after disinfection and 10 consecutive days after disinfection. Bacteria in the water samples were cultured for the colony counts. Then, the bacterial culture data were compared between groups. The qualified criterion of the water sample was that the number of bacterial colonies was less than or equal to 100 CFU/mL. After disinfection, a bacterial species mass spectrometry identification analysis was carried out when the number of bacterial colonies in each group exceeded the standard for the first time. Biofilms from the inner wall of the tube in the three groups were observed under an electron microscope on the 10th day after disinfection. Results There were no significant differences between the three groups before disinfection (F = 2.549, P = 0.111). The number of bacterial colonies in the spout of 6 scalers in the control group all exceeded the standard, and three kinds of bacteria were cultured: Sphingomonas melonis, Herbaspirillum huttiense, and Ralstonia pickettii. Compared with those in the control group, the number of bacterial colonies in experimental group 1 decreased significantly for 1-2 days after disinfection (P