Objective: In this in vitro study the antibacterial substantivity of a new sodium hypochlorite-based root canal irrigant (Hypoclean) in bovine root dentin was investigated. Study Design: Ninety dentin tubes prepared from bovine incisor teeth were used. After contamination for 14 days with Enterococcus faecalis, the specimens were divided into five groups as follows: Hypoclean; Tetraclean; 5.25% sodium hypochlorite (NaOCl); infected dentin tubes (positive control); and sterile dentin tubes (negative control). Dentin chips were collected with round burs into tryptic soy broth and after culturing, the number of colony-forming units (CFU) was counted. Results: The number of CFU was minimum in the first cultures in all experimental groups, and the results obtained were significantly different from each other at any time period (P < 0.05). At all five experimental periods, the Tetraclean group showed the most effective antibacterial action (P < 0.05). Regular 5.25% NaOCl group showed the worst result at all periods. Hypoclean group demonstrated no bacterial growth after treatment. In each group, the number of CFU increased significantly by time-lapse (P < 0.05). Conclusion: The residual antibacterial activity of Tetraclean was significantly greater than Hypoclean and 5.25% NaOCl. © Medicina Oral S. L.