OBJECTIVE: Oral biofilm has a high acid-producing capacity, increases the risk of enamel demineralization around brackets, and has been identified as a problem in orthodontic treatment. Here, we assessed the risk of biofilm formation by Streptococcus mutans, which is associated with the development of white spot lesions (WSL) on tooth surfaces, using multibracket devices. METHODS: Various types of brackets were used for the biofilm formation assay with S. mutans coated with human saliva, immersed in renG-expressing S. mutans UA159 (strain with the luciferase gene inserted), and incubated overnight at 37 °C under aerobic conditions containing 5% CO RESULTS: Biofilm formation was greater in plastic brackets than in ceramic and metal brackets in a number-dependent manner. However, biofilm formation was inhibited as the plastic bracket was coated with saliva. CONCLUSION: For preventive treatments of WSL onset during orthodontic treatment, orthodontists should carefully select and customize brackets based on patient needs, goals, and biomechanical principles. This study developed a new measurement method using renG-expressing S. mutans UA159 to accurately assess active biofilm formation on bracket surfaces.