Bacteria remaining within the root canal system are a significant factor in endodontic failures. The aim of this in vitro study was to evaluate the antimicrobial efficacy of commercially available gutta-percha formulations against common endodontic pathogens. Standard, tetracycline, or iodoform (also called medicated gutta-percha; MGP) containing gutta-percha were tested against Actinomyces israelii, A. naeslundii, Enterococcus faecalis, and Fusobacterium nucleatum using the agar diffusion method. Resilon points and tetracycline disks/E-tests were used as negative and positive controls, respectively. Resilon did not display antimicrobial properties. Standard gutta-percha and MGP inhibited F. nucleatum and A. naeslundii, with MGP also inhibiting A. israelii. Tetracycline containing gutta-percha was found to inhibit growth from all four bacterial species tested. The results of this study suggest that the use of tetracycline containing gutta-percha as an obturation material may be useful as a complement to the cleaning and disinfection phase of root canal procedures.