Several dye leakage studies have demonstrated the fact that mineral trioxide aggregate (MTA) leaks significantly less than other root-end filling materials. The purpose of this study was to determine the time needed for Serratia marcescens to penetrate a 3 mm thickness of zinc-free amalgam, Intermediate Restorative Material (IRM), Super-EBA, and MTA when these materials were used as root-end filling materials. Fifty-six, single-rooted extracted human teeth were cleaned and shaped with a series of .04 Taper rotary instruments (Pro-series 29 files). Once the canals were prepared in a crown down approach, the ends were resected and 48 root-end cavities were ultrasonically prepared to a 3 mm depth. The teeth were then steam sterilized. Using an aseptic technique, under a laminar air flow hood, the root-end cavities were filled with amalgam, IRM, Super-EBA, and MTA. Four root-end cavities were filled with thermoplasticized gutta-percha without a root canal sealer and served as positive controls. Another four root-end cavities were filled with sticky wax covered with two layers of nail polish and served as negative controls. The teeth were attached to presterilized (ethylene oxide gas) plastic caps, and the root ends were placed into 12-ml vials of phenol red broth. Using a micropipette, a tenth of a milliliter of S. marcescens was placed into the root canal of each tooth. To test the sterility of the apparatus set-up, the root canals of two teeth with test root-end filling materials and one tooth from the positive and negative control groups were filled with sterile saline. The number of days required for S. marcescens to penetrate the four root-end filling materials and grow in the phenol red broth was recorded and analyzed. Most of the samples filled with zinc-free amalgam leaked bacteria in 10 to 63 days. IRM began leaking 28 to 91 days. Super-EBA began leaking 42 to 101 days. MTA did not begin leaking until day 49. At the end of the study, four of the MTA samples had not exhibited any leakage. Statistical analysis of the data indicated Mineral Trioxide Aggregate to be a most effective root-end filling material against penetration of S. marcescens.