The aim of this study was to evaluate the residual antibacterial activity of Tetraclean, MTAD and 5.25% sodium hypochlorite (NaOCl) in bovine root dentin in vitro. One hundred and ten dentin tubes prepared from bovine incisor teeth were infected in vitro for 14 days with Enterococcus faecalis. Thereafter, the specimens were divided into five groups as follows: Tetraclean; MTAD; 5.25% NaOCl; infected dentin tubes (positive control); and sterile dentin tubes (negative control). Dentin chips were collected using round burs into tryptic soy broth and after culturing, the number of colony-forming units (CFU) was counted. The number of CFU in all experimental groups was minimum after treatment, and the results obtained were significantly different from each other at any time period (P < 0.05). The Tetraclean group showed the most effective antibacterial action at all five experimental periods (P < 0.05). MTAD group showed the least antibacterial activity after treatment. However, at days 7, 14, 21 and 28 MTAD showed more effective antibacterial action than NaOCl. In each group, the number of CFU increased significantly by time-lapse (P < 0.05). In conclusion, the residual antibacterial activity of Tetraclean was significantly greater than MTAD and 5.25% NaOCl.