Aim: To evaluate the effectiveness of different type of medicated and non-medicated gutta-percha points in reducing bacterial colonization in root canal system. Methodology: Three different bacterial strains were used Staphylococcus aureus, Escherichia Coli, Enterococcus Faecalis .Disc was prepared from Whatmann filter paper. Then with the help of tuberculin syringe, 1 ml of the specific solution of gutta-percha points, was added on each disc separately. Then it was allowed to dry at room temperature to avoid excessive moisture accumulation and stored at 2 to 8˚C. Zones of inhibition were measured to nearest mm, and the results were interpreted with the assistance of diameter. Result: On the basis of zone of inhibition, it was maximum in group IV (Chlorhexidine GP) followed by group III (Calcium hydroxide GP) than group II (conventional GP).Conclusion: Roeko activ (chlorhexidine gutta-percha points) showed higher antimicrobial efficacy than Calcium hydroxide gutta-percha points against the microorganisms tested.