The purpose of this study was to investigate the efficacy of chlorhexidine (CHX) and calcium hydroxide (Ca(OH2) against Enterococcus faecalis in vitro. Extracted single-rooted human teeth were instrumented up to size 40. After removal of the smear layer, an inoculum of E. faecalis was inserted into the root canals. After incubation, the inoculum was removed and the root canals were filled with one of three different disinfectants: Ca(OH2 paste, CHX 2%, and a mixture of CHX and Ca(OH2 paste (n = 10 in each group). Control teeth were filled with water of standardized hardness (n = 10). The teeth were then incubated for 3 days. After incubation, each root canal was instrumented, and the removed dentin was examined microbiologically. CHX was significantly more effective against E. faecalis than was Ca(OH2 paste or a mixture of CHX with Ca(OH2 paste (p < 0.05). There was no increase in the efficiency of Ca(OH2 paste when CHX was added (p > 0.05). The results suggest that CHX is effective in the elimination of E. faecalis from dentinal tubules under the conditions of this study.