The purpose of this investigation was to compare the antimicrobial effect of MTAD (a mixture of a tetracycline isomer, an acid, and a detergent) with that of NaOCl with and without EDTA. Eighty-five extracted human teeth were contaminated with Enterococcus faecalis for 4 weeks. After biomechanical instrumentation using 1.3% or 5.25% NaOCl as root canal irrigant, the root canal and the external surface of each tooth were exposed to a 5-min application of MTAD, 1.3% NaOCl, 5.25% NaOCl or a 1-min application of EDTA followed by irrigation with 5 ml of 1.3% NaOCl or 5.25% NaOCl. Teeth or dentin shavings were cultured to determine presence or absence of the test bacteria. Fisher's exact test showed that the combination of 1.3% NaOCl as a root canal irrigant and MTAD as a final rinse was significantly more effective against E. faecalis than the other regimens. The chi2 test showed no difference between the other regimens.