Introduction: One of the important issues in root canal treatment is the elimination of the microorganisms in the root canal before obturation. This study aimed to experimentally investigate the combined effects of ethanol extract of Allium hirtifolium (shallot) and calcium hydroxide on the biofilm structure of the resistant bacterium Enterococcus faecalis. Method: Dried Allium hirtifolium was extracted with ethanol. Various concentrations (0-20-40-50-60-80-100 mg/ml) of the extract and calcium hydroxide were prepared. Antibacterial effects of these compounds were evaluated against Enterococcus faecalis using disc-diffusion, well-diffusion, and biofilm formation inhibition assays, along with optical absorption measurements. Results: The results showed that in disc-diffusion and well-diffusion methods, the highest antibacterial effects were observed in the positive control group (antibiotics amoxicillin and co-amoxiclav), 100 mg/ml calcium hydroxide, and combined concentrations of calcium hydroxide and Allium hirtifolium (80/20, 60/40 and 50/50 mg/ml). Other concentrations of these compounds did not affect Enterococcus faecalis. Regarding biofilm formation, the study revealed that the ability of Enterococcus faecalis to form biofilm was weak at concentrations of 100, 80, and 60 mg/ml of calcium hydroxide, while other concentrations of the studied substances showed average inhibition. Conclusion: The present study revealed that in disc diffusion and well-diffusion methods, ethanol extract of Allium hirtifolium alone or in combination with calcium hydroxide did not significantly inhibit the growth of Enterococcus faecalis, while calcium hydroxide exhibited the highest (100%) antibacterial effect
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