Introduction: Endodontic infection in primary teeth is a multimicrobial disease involving a rich combination of bacterial species. The secondary metabolites from the medicinal plant Phyllanthus niruri L. have antimicrobial properties. This in vitro research evaluated the antimicrobial activity of P. niruri and its mode of action against Enterococcus faecalis, Porphyromonas gingivalis, and Treponema denticola. Methodology: The antimicrobial activities of an ethanolic extract of P. niruri and a triple antibiotic paste (TAP) as a positive control were evaluated using the disc diffusion method, and their minimum inhibitory concentration (MIC) and minimum bacterial concentration were determined. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the cellular damage induced by the extract. A one-way ANOVA followed by Tukey’s post hoc test was used to compare the mean zone of inhibition for all three microbes. The level of significance was set at P < 0.05. Results: The herbal extract of P. niruri had the highest inhibitory effects against T. denticola (16.06 ± 3.13), followed by P. gingivalis (14.54 ± 2.28 mm), and E. faecalis (10.10 ± 0.71 mm). However, it was less than the TAP ( p < 0.001). P. niruri exhibited bacteriostatic potential with its MIC against all microbes. Furthermore, SEM and TEM found severe membrane deformation and extensive cytoplasmic leakage in T. denticola and P. gingivalis compared to E. faecalis. Conclusion: Based on the study results, a 250 mg/mL concentration of P. niruri could be beneficial as a potential medicament for reducing root canal pathogens.
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