Objective: To evaluate and compare the antibacterial efficacy of 10% copaiba oil and 5.25% sodium hypochlorite in disinfecting gutta-percha cones experimentally contaminated with Enterococcus faecalis, Streptococcus mutans, and Streptococcus oralis.

Materials and Methods: An in vitro comparative experimental study was conducted using 180 gutta-percha cones, randomly assigned to nine experimental subgroups (n = 20 each). The cones were contaminated with standardized bacterial strains and subsequently treated with 10% copaiba oil, 5.25% sodium hypochlorite, or distilled water (negative control). Antibacterial activity was assessed by counting colony-forming units (CFU). Data normality was tested using the Shapiro–Wilk test, and intergroup comparisons were performed with the Kruskal–Wallis test followed by Dunn’s post hoc test, with a significance threshold of p < 0.05.

Results: Sodium hypochlorite exhibited the greatest antibacterial efficacy against all three bacterial species, with mean counts of 0.8 ± 2 CFU for E. faecalis, 1.4 ± 2.3 CFU for S. mutans, and complete inhibition of S. oralis. Copaiba oil also significantly reduced bacterial growth compared with the negative control. Statistically significant differences were observed among the three treatments for all bacterial species (p < 0.005).

Conclusions: 5.25% sodium hypochlorite demonstrated the highest antibacterial efficacy; however, 10% copaiba oil showed notable antimicrobial activity, particularly against S. oralis, suggesting its potential as a natural alternative and warranting further investigation in endodontics.