Objective: The antibacterial and antifungal effects of medicated gutta-percha (MGP) over different time periods have not been evaluated. The aim of this study was to evaluate and compare the antimicrobial and antifungal effectiveness of MGP and regular gutta-percha cones over different time periods using the disk diffusion method. Methods: Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli were spread onto Mueller-Hinton agar, and Candida albicans was spread onto Sabouraud agar supplemented with glucose. Same-size MGP cones, conventional gutta-percha cones and disks impregnated with povidone-iodine were placed on the inoculated plates. The dishes were incubated at 37°C aerobically. Growth inhibition zones were inspected and measured at 24, 48 and 72 hours. For each strain, experiments were performed in triplicate. The Kruskal-Wallis test was used to compare k independent samples. Results: The disks impregnated with povidone-iodine inhibited all bacterial strains for up to 72 hours. No inhibition zones were seen around regular gutta-percha cones. MGP inhibited the growth of all bacteria over 24 hours, but in some cases these effects did not continue over longer periods. Specifically, the antimicrobial properties of MGP cones against E. coli and P. aeruginosa disappeared by 48 and 72 hours. Regardless of time and bacterial strain, MGP was statistically more effective than regular guttapercha (p < 0.05). Povidone-iodine and MGP were effective against C. albicans for up to 72 hours, but regular gutta-percha exhibited no antifungal activity. Conclusion: The antimicrobial and antifungal characteristics of MGP may offer additional advantages over conventional gutta-percha. © J Can Dent Assoc 2006.