Objective: To investigate the effect of the roselle calyx extract (RCE) (Hibiscus sabdariffa L.) on the in vitro viability and biofilm formation ability of oral pathogenic bacteria. Methods: RCE was prepared by soaking roselle calyx powder with ethyl alcohol for 24 h at room temperature. After centrifugation, the extract was lyophilized. Then, the extract was dissolved in phosphate-buffered saline, the pH was adjusted, and the extract was aseptically filtered. We used Streptococcus mutans, Streptococcus sanguinis, Lactobacillus casei, Actinomyces naeslundii, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia in this study. The antibacterial activity of the RCE was determined by treating the cells of these bacteria with the extract for 10 or 20 min at room temperature. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration was determined using the microdilution method, and the effect of the RCE on the ability to form biofilm was determined using a polystyrene micro plate assay. In addition, we used the WST-1 assay to determine the cytotoxicity of the RCE on HGF, Ca9-22 and KB cells. Results: The RCE had antibacterial activity against oral bacteria used in this study. In particular, most significant antibacterial activity was observed against Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis. The MIC and minimum bactericidal concentration were 7.2 mg/mL-28.8 mg/mL and 14.4 to >57.6 mg/mL. The RCE had an inhibitory effect on biofilm formation at the MIC and sub-MIC levels. In addition, the RCE had low cytotoxic effects on HGF, Ca9-22 and KB cells. Conclusions: Thus, our results indicate that the RCE may be used for preventing oral diseases.