The purpose of this study was to evaluate the antimicrobial effects of a toothbrush with light-emitting diodes (LEDs) on periodontitis-associated dental biofilm attached to a zirconia surface by static and dynamic methods. Materials and Methods: Zirconia disks (12 mm diameter, 2.5 mm thickness) were inserted into a 24-well plate (static method) or inside a Center for Disease Control and Prevention (CDC) biofilm reactor (dynamic method) to form dental biofilms using Streptococcus gordonii and Fusobacterium nucleatum. The disks with biofilm were subdivided into five treatment groups-control, commercial photodynamic therapy (PDT), toothbrush alone (B), brush with LED (BL), and brush with LED+erythrosine (BLE). After treatment, the disks were agitated to detach the bacteria, and the resulting solutions were spread directly on selective agar. The number of viable bacteria and percentage of bacterial reduction were determined from colony counts. Scanning electron microscopy (SEM) was performed to visualize alterations in bacterial morphology. Results: No significant difference in biofilm formation was observed between dynamic and static methods. A significant difference was observed in the number of viable bacteria between the control and all experimental groups (P < 0.05). The percentage of bacterial reduction in the BLE group was significantly higher than in the other treated groups (P < 0.05). SEM revealed damaged bacterial cell walls in the PDT, BL, and BLE groups, but intact cell walls in the control and B groups. Conclusion: The findings suggest that an LED toothbrush with erythrosine is more effective than other treatments in reducing the viability of periodontitis-associated bacteria attached to zirconia in vitro.