AimThe aim of this study is to compare and evaluate the antimicrobial efficacy of chlorhexidine, calcium hydroxide, and cetylpyridinium chloride againstEnterococcus faecalisin the presence and absence of contamination with simulated periapical exudate at different time intervals.MethodsSimulated wound exudate and cetylpyridinium chloride gel were prepared prior to testing. The test groups were divided into groups A and B based on the presence and absence of simulated wound exudate. They were further divided into four subgroups as follows: subgroup 1: calcium hydroxide; subgroup 2: 2% chlorhexidine gel; subgroup 3: 0.5% cetylpyridinium chloride gel; subgroup 4: 0.9% saline as control.E. faecaliswas inoculated, and the test groups were evaluated at different time periods of six, 12, and 24 hours. Aliquots were then obtained and subjected to 10-fold serial dilutions. A total of 10 µl of individual samples was spread onto the nutrient agar medium using L-rod. The plates were then assessed for colony-forming units (CFU), and the values obtained were subjected to statistical analysis. Kolmogorov-Smirnov and Shapiro-Wilk normality tests were used to check whether the variables follow a normal distribution. For within-group comparison, the Friedman test and the Kruskal-Wallis test were used. For between-group comparison, the Mann-Whitney U test was used.ResultsSaline had the highest CFU values, while cetylpyridinium chloride had the lowest CFU values in both contaminated and non-contaminated groups. In all the conditions, the CFU values of cetylpyridinium chloride were significantly lowest compared to the other three groups. CFU values of the calcium hydroxide group were significantly high, followed by the chlorhexidine group when compared to cetylpyridinium chloride in both contaminated and non-contaminated groups.ConclusionWithin the limitations of the current study, it can be concluded that cetylpyridinium chloride was the most effective intracanal medicament againstE. faecalisthan calcium hydroxide and chlorhexidine at varying time intervals, even in the presence of a periapical exudate. Thus, cetylpyridinium chloride can be considered an effective intracanal medicament for root canal disinfection.