Endodontic treatment failure can be caused by Gram-positive bacteria, Enterococcus faecalis. Natural ingredients such as beetroot extract (Beta vulgaris L.) can be used to inhibit the growth of Gram positive bacteria. Beetroot extract (Beta vulgaris L.) has antibacterial properties because it contains active substances of flavonoids, phenols, tannins, saponins and alkaloids.To determine the antibacterial effect of beetroot extract (Beta vulgaris L.) against Enterococcus faecalis bacteria.This study was a quasi-experimental study consisting of five treatment groups, namely the beetroot extract group with concentrations of 10,000 µg/ml, 12,500 µg/ml, and 15,000 µg/ml, and 2% Chlorhexidine Gluconate as positive control, and Aquadest as negative control. Test the antibacterial effect using the well-diffusion method with Mueller Hinton Agar media. The repetition of the test for each group was 3 times. The media was incubated for 24 hours at 37⁰C. After 24 hours, the diameter of the inhibition zone was calculated using a caliper in millimeters. Data were analyzed with the Kruskal-Wallis test and the Mann-Whitney test.There was a significant difference between all treatment groups (p<0.05). Only beetroot extract concentration of 10,000 µg/ml did not show an inhibition zone, while concentrations of 12,500 µg/ml and 15,000 µg/ml showed an inhibition zone. The diameter of the largest inhibition zone was at a concentration of 12,500 µg/ml, which was 2.33 ± 0.06 mm, but still smaller than the positive control, namely Chlorhexidine Gluconate 2%, which was 2.33 ± 0.06 mm.Beetroot extract (Beta vulgaris L.) can inhibit the growth of Enterococcus faecalis bacteria with the largest inhibition zone diameter achieved by a concentration of 15,000 µg/ml