Aims: To evaluate the effectiveness of grape seed extract (GSE) as a gutta-percha disinfectant and to compare the efficacy with 5% sodium hypochlorite, 2.5% sodium hypochlorite, and 2% chlorhexidine against Bacillus subtilis.Materials and methods: Minimum inhibitory concentration and minimum bactericidal concentration of GSE were calculated by serial dilution and well-diffusion method. Five hundred gutta-percha cones of ISO size 25 were divided into 10 equal groups (n = 50). The 10 groups of gutta-percha cones were inoculated with cultured Bacillus species and incubated for 72 hours for allowing bacterial growth. Each group was then treated with the test solutions for 1 and 5 minutes. The treated groups of gutta-percha cones were then incubated in brain heart infusion agar allowing for bacterial growth, which were later analyzed by the turbidity of the medium. The results were statistically analyzed.Results: Gutta-percha cones when treated with 2% chlorhexidine gluconate for 1 and 5 minutes showed the most inhibiting activity against B. subtilis. Grape seed extract was found to have limited activity against B. subtilis in both 1-and 5-minute interval. Both concentrations of NaOCl, 2.5 and 5%, showed reduced activity against B. subtilis. Conclusion:Grape seed extract though has antibacterial activity, when used as gutta-percha disinfectant, was found to be less effective than chlorhexidine gluconate against B. subtilis.Clinical significance: Different methods of gutta-percha cone disinfection have been advocated and GSE for gutta-percha cone disinfection was attempted owing to its herbal antibacterial nature.