Background: Perforations either pathologic or iatrogenic can result in inflammatory reaction to the periodontium, bone resorption, and the formation of granulomatous tissue. If not sealed properly, reinfection in the root canal space can occur leading to periapical lesions, bone loss and eventually tooth loss. Objectives: To evaluate the antimicrobial effect of nano-silver and nano-chitosan impregnated MTA compared to conventional MTA by confocal scanning laser microscopy. Materials and Methods: Forty eight samples were divided into three groups according to the tested material: group A was nanosilver impregnated MTA, group B was nanochitosan impregnated MTA and group C was conventional MTA. After bacterial inoculation to the dentin blocks and the addition of the tested materials to each dentin specimen, the samples were stained by Acridine orange and Proprium iodine. Image analysis was done using Zeiss blue software to count the number of dead and living bacterial cells. The percentage of living bacteria was calculated by dividing the number of live bacteria by the total number of bacteria then multiplied by hundred. Results: Nanosilver impregnated MTA showed a significantly higher antibacterial property followed by the nanochitosan impregnated MTA and finally the conventional MTA. Conclusion: The antibacterial property and solubility of MTA was enhanced by the addition of nanoparticles. The addition of Nanosilver to MTA presented a greater antibacterial effect than nanochitosan.