Aim: To demonstrate the effectiveness of disinfecting substances with 2% and 5% Sodium Hypochlorite and 2% Chlorhexidine Gluconate at each of the pre-established times of 0:15 and 0:30 seconds, and 1, 2, 5, and 10 minutes. Methods: This study selected 96 gutta-percha cones that were contaminated with Enterococcus Faecalis, dried and treated with the aforementioned substances and applied at pre-established times. Subsequently, these were transferred to sterile Brain Heart Infusion broth and placed in a bacteriological incubator at 37°C for 24 hours to evaluate microbial growth, as well as in a nutrient agar medium in Petri dishes. Half of the cone was transferred to individual filter paper packages and exposed to the environment in a dental clinic at Universidade José do Rosário Velano, for 7 days, with subsequent evaluation for microbial growth. The bacterial phenotype test was performed using Gram stain and growth in 6.5% saline solution. The results were submitted to statistical analysis using the Kruskal Wallis H test, with a significance level of 5%. Results: The substances were effective at all times tested and individual storage supported disinfection. In the statistics test, the p-value was greater than 0.05, as there was no variability in the data configurations. Conclusion: The disinfection of gutta-percha cones and individual storage was an effective protocol to be adopted with 2% and 5% Sodium Hypochlorite and 2% Chlorhexidine.