Introduction The main aim of this study was to compare and evaluate different chemical agents and herbal products in disinfecting gutta-percha (GP) cones through an in vitro study. Materials and Methods For this study, 160 GP cones were taken in test tubes, which were contaminated with Enterococcus faecalis inocula, prepared by dissolving it in Brain Heart Infusion (BHI) broth. The GP cones were then taken from the test tubes and dried on Whatman filter paper no. 1 and were divided into four groups according to the decontaminant used for the study—group 1 (5% sodium hypochlorite [NaOCl]), group 2 (2% chlorhexidine), group 3 (propolis), and group 4 (Aloe vera)—which were further subdivided into subgroups on the basis immersion time periods of 1 and 10 minutes. The GP cones were then again dried and taken in the test tubes containing freshly prepared BHI broth to check the turbidity. Results It was found that 2% chlorhexidine was the most effective disinfectant against E. faecalis, 5% NaOCl was the second best disinfectant followed by propolis, while Aloe vera had not shown any effect as GP disinfectant. Conclusion Since 2% chlorhexidine showed better disinfection efficacy against E. faecalis than 5% NaOCl, it can be recommended for chairside disinfection of GP. One-minute immersion of GP in 2% chlorhexidine is sufficient for elimination of E. faecalis.