Most dental and gingival pathology were triggered by oral biofilm which consisted ofStreptococcus sanguinis as an early colonizer. S. sanguinis could elicit some pathological events such as dental caries, periodontal diseases. One of the strategies to achieve such result is with antimicrobial therapy. The widely known gold standard is the utilization of chlorhexidine which has also been known to discolor the tongue and dentition, also change in sensation and mucosal irritation. Those disadvantages have resulted in some research toward the therapy from natural resources, such as the calyx of Hibiscus sabdariffa. S. sanguinis between 20 and 24 h in biofilm was exposed to the ethanol extracts of H. sabdariffa calyx at the concentration of 0.2% to 50% for 60 min. Subsequently, the cellular viability was tested with crystal violet assay. As the positive control group, chlorhexidine 0.1% and 0.2% were used. The viability was expressed in percents toward the negative controls. The results were analyzed with one way ANOVA (P<0.05). The viability of S. sanguinis was tending to decrease as the concentration increases. The ethanol extract ofH. sabdariffa calyx is capable of reducing the S.sanguinis viability in the biofilm, where the effectiveness of the ethanol extract of H. sabdariffa calyx as antibacterial agent was affected by biofilm formation phase. The effectiveness of the ethanol extract of H. sabdariffacalyx in reducing S. sanguinis viability is equal to chlorhexidine, at the adherence phase (20 h) and maturated phase (24 h) of biofilm. Key words: Ethanol extract, Hibiscus sabdariffa calyx, Streptococcus sanguinis,chlorhexidine, Biofilm, Viability.