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This in vitro study aimed to evaluate the growth-inhibitory effects against periodontal disease-causing bacteria and cytotoxic effects against mouse fibroblast cells of the Stryphnodendron adstringens (barbatimão) hydroalcoholic extract. The contents of phenols and tannins in the extract were determined. The growth-inhibitory activity of the barbatimão was evaluated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The viability of fibroblast cells was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assay at 24 and 48 h post-treatment. The MIC values of the extract against Prevotella intermedia, Porphyromonas gingivalis, and Fusobacterium nucleatum were 0.05, 0.125, and 2 mg mL-1, respectively, while the MBC values were 4, 2, and 2 mg mL-1, respectively. The viability rate of barbatimão (0.25 mg mL-1)-treated L929 cells was higher than that of chlorhexidine (0.12%)-treated L929 cells at 48 h post-treatment. The contents of total phenolics and total tannins in the extract were 837.39 ± 0.10 and 785.82 ± 0.14 mg of tannic acid equivalent per gram of the extract, respectively. These findings indicate that the barbatimão hydroalcoholic extract, which exerted potent growth-inhibitory effects against the test microbial species and low cytotoxic effects on fibroblasts, has potential applications in the development of novel mouthwash products., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)