Objective: This study aimed to investigate the antimicrobial efficacy of propolis against key endodontic pathogens, such as Enterococcus faecalis, Staphylococcus aureus, and Candida albicans. The primary focus was on determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) of the ethanolic extract of propolis. Methods: Raw propolis was obtained from beekeepers in Chennai, India, and subjected to maceration. The ethanolic extract was then evaluated for its effectiveness against the specified pathogens through conventional broth dilution techniques. Bacterial and fungal concentrations were adjusted to standardized levels, and MIC and MBC/MFC values were determined through serial dilutions. Statistical analysis, including ANOVA and Tukey's post hoc test, was utilized to evaluate the significance of the results. Results: Propolis exhibited concentration-dependent antimicrobial effects. For Staphylococcus aureus, the MIC and MBC were determined to be 20 mg/ml. Similarly, Candida albicans showed a MIC and MFC of 20 mg/ml. Enterococcus faecalis displayed susceptibility to the ethanolic extract of propolis, with an MBC of 5 mg/mL. Conclusion: The concentration-dependent antimicrobial effectiveness of the ethanolic extract of propolis against specific bacteria and fungi provides a promising avenue for enhancing infection control in endodontics. Despite these positive results, further research and clinical trials are necessary to establish optimal application protocols and ensure the safety of propolis as an intracanal medicament.