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The main aim of this study was to determine and compare the antimicrobial effect of hibiscus acid and a commercial 0.12% (w/v) chlorhexidine mouthrinse against Streptococcus mutans, Streptococcus sanguinis, Capnocytophaga gingivalis, and Staphylococcus aureus, and to determine the effect on bacterial cell membrane permeability and the toxicity of hibiscus acid in a mouse model. Hibiscus acid was obtained from acetone extract of Hibiscus sabdariffa calyces. Chlorhexidine (0.12% w/v) mouthrinse was purchased from a local pharmacy. The antimicrobial activity of hibiscus acid and mouthrinse were determined using the gel diffusion technique. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the solutions were determined using the broth dilution method. The effect on bacterial cell membrane permeability of hibiscus acid and mouthrinse was determined by crystal violet assay. The toxicity of hibiscus acid was investigated in a mouse model (registration number: UAEH2019-A1-S-8288). Hibiscus acid and mouthrinse showed antibacterial activity against all oral pathogenic bacteria. However, hibiscus acid showed a lower antibacterial effect compared with chlorhexidine mouthrinse. The MIC and MBC for hibiscus acid were 3 and 5 mg/mL, respectively, and was between 30 and 50 μg/mL for mouthrinse. The crystal violet test results indicate that hibiscus acid and mouthrinse alter the permeability of the bacterial membrane. Finally, hibiscus acid did not show toxicity in mouse studies. [ABSTRACT FROM AUTHOR]