In the present study, the residual antibacterial activity, or substantivity, of three concentrations of Tetraclean (Ogna Laboratori Farmaceutici, Muggiò, Italy) was assessed in bovine root dentin in vitro. One hundred 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: 100% Tetraclean, 10% Tetraclean, 1% Tetraclean, sterile dentin tubes (negative control), and infected dentin tubes (positive control). Dentin chips were collected with round burs into tryptic soy broth and, after culturing, the number of colony-forming units (CFU) was counted. The number of CFU was minimal in the first cultures in all experimental groups, and the results obtained were significantly different in the different groups at all time periods (p<0.05). At all five experimental periods, the 100% Tetraclean group showed the most effective antibacterial action (p<0.05). In each group, the number of CFU increased significantly with time (p<0.05). There was a direct relationship between the concentration of Tetraclean and its substantivity. In conclusion, under the conditions of the study presented here, the substantivity of 100% Tetraclean was significantly higher than that of lower concentrations.