B ackground: Periodontitis, one of the major causes of tooth loss, is commonly found in society which initiated by the accumulation of biofilm on the tooth surface. Porphyromonas gingivalis and Treponema denticola are bacteria that play a crucial role in periodontitis. Hibiscus sabdariffa L. contains flavonoids, alkaloids, saponins, and tannins compounds which are expected to inhibit biofilm formation with minimal side effects. Objectives: To determine the effect of H. sabdariffa L. extract on the formation of monospecies T. denticola and multispecies P. gingivalis and T. denticola biofilms. Methods: In vitro experimental laboratory using post-test only control group design with the biofilm assay method. This study used H. sabdariffa L. calyxes extract with concentrations of 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, and 1.5625%, 0.2% chlorhexdine as positive control, and BHI as negative control. The extracts were distributed into well-plates and incubated for 1, 6, and 24 hours. The microplate reader is used to read the optical density of the biofilm. Results: H. sabdariffa L. calyx extract starting from a concentration of 1.5625% at an incubation period of 1 hour and 24 hours, and extracts starting from a concentration of 3.125% at an incubation period of 6 hours were able to inhibit the formation of monospecies T. denticola biofilms more effectively than chlorhexidine (p<0.05). Extracts starting at a concentration of 1.5625% at an incubation period of 1 hour, extracts starting at a concentration of 6.25% at an incubation period of 6 hours, and extracts starting at a concentration of 3.125% at an incubation period of 24 hours are able to inhibit the formation of multispecies biofilms better than chlorhexidine (p<0.05). Conclusion: H. sabdariffa L. calyxes extract significantly inhibits P. gingivalis and T. denticola, either as monospecies or multispecies biofilm.