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Aims and Objectives: The aims of the present study were to determine and compare the effects of different concentrations of Althaea officinalis extract, 0.2% chlorhexidine (CHX), and penicillin on Streptococcus mutans and Lactobacillus acidophilus in vitro. Materials and Methods: The laboratory study was done, for a period of 8 weeks. Minimum inhibitory concentration (MIC) in the test tube, minimum bactericidal concentration (MBC) in a plate culture medium, and growth inhibition zone diameter methods were used to compare the antibacterial effects of 0.2% CHX, penicillin, and different concentrations of A. officinalis root extract. The data were analyzed by SPSS version 24 using ANOVA and t-test analysis. Results: The results showed A. officinalis root extract had antibacterial effect, but significant differences were in MIC and MBC against L. acidophilus and S. mutans with penicillin and 0.2% CHX mouthwash. In addition, the mean growth inhibition zones of all the concentrations of the plant extract were less than that of the positive control group (P = 0.001). However, the difference in the maximum growth inhibition zone from that with the negative control group was significant. In addition, the antibacterial effect of the extract increased with an increase in its concentration. The extract exerted a greater antibacterial effect on S. mutans than on L. acidophilus. The plant polyphenols content is 3.7% which is equivalent to 29.93 g/ml. Conclusion: The root extract of A. officinalis exhibited antibacterial effects on S. mutans and L. acidophilus, but this effect was less than those of CHX mouthwash and penicillin. The antibacterial effect increased with an increase in the concentration of the extract. [ABSTRACT FROM AUTHOR]