B Background: Periodontal disease is a disease that has a high prevalence globally and in Indonesia. There are 2 types of periodontal disease, such as gingivitis and periodontitis which can be caused by pathogenic bacteria such as Aggregatibacter actinomycetemcomitans dan Fusobacterium nucleatum. Both of these bacteria are Gram-negative that are closely related to the occurrence of periodontal disease. To minimalize periodontal disease can be done by brushing the teeth, scaling, flossing, and using mouthwash that contains antibacterial. Rosella (Hibiscus sabdariffa L.) is an herbal plant that contains antibacterial properties, so it can be used as an alternative to minimize periodontal disease. Methods: The experiment was held at the Micore FKG Usakti laboratory with an in vitro experimental laboratory type using biofilm assay tests with concentrations of 1 g/mL, 0,50 g/mL, 0,0.25g/mL, 0,125g/mL, 0,0625 g/mL, 0,0312 g/mL, 0,015 g/mL. The extract was distributed into well plates and then stained with crystal violet and incubated for 1 hour, 6 hours and 24 hours and then counted using a microplate reader. Results: Rosella extract can inhibit the growth of multispecies bacteria Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum. Extract concentrations starting from 0.015 g/mL at an incubation period of 1 hour, concentrations starting at 0.125 g/mL at an incubation period of 6 hours, and concentrations starting from 0.0625 g/mL at an incubation period of 24 hours of incubation period were proven to inhibit bacterial growth and proved to be more effective than 0.2% chlorhexidine. Conclusion: Rosella extract can inhibit the growth of multispecies bacteria Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum.