To develop a root canal filling material with high antimicrobial activity, we prepared gutta-percha supplemented with the cationic surfactant cetylpyridinium chloride (CPC). Thermoplastic gutta-percha was supplemented with 0.05%, 0.2%, or 0.8% CPC. The gutta-percha containing CPC was tightly packed at the bottom of a 24-well plate. Its antimicrobial activity against eight representative endodontic pathogens-including gram-positive and gram-negative bacteria and fungi-was evaluated by adding 0.5 mL of liquid samples containing pathogens to the wells. After 24 h of cultivation under appropriate conditions, microbial growth was analyzed by counting colony-forming units (CFU). Gutta-percha alone (without CPC) partially inhibited microbial growth, probably through the antimicrobial effect of some of its components, such as zinc oxide. Addition of CPC dose-dependently increased the antimicrobial efficacy of gutta-percha. Addition of 0.05%, 0.2%, and 0.8% CPC reduced the viable microbial number to below the lower limit of detection (20 CFU/mL) for all tested pathogens except Pseudomonas aeruginosa, which was detected in 0.8% CPC-containing gutta-percha, although the viable number significantly decreased. Gutta-percha with CPC might be useful for preventing microbial infections during root canal therapy. (J Oral Sci 58, 277-282, 2016).