The aim of this in vitro study was to compare the antimicrobial substantivity of 2% chlorhexidine gluconate (CHX), 100 mg ml(-1) doxycycline and 2.6% sodium hypochlorite (NaOCl) in bovine root dentine. Eighty dentine tubes prepared from bovine incisors were infected in vitro for 14 days with Enterococcus faecalis. The specimens were divided into five groups as follows: doxycycline HCl; CHX; NaOCl; infected dentine tubes (positive control); and sterile dentine tubes (negative control). Dentine chips were collected with round burs into tryptic soy broth. After culturing, the number of colony-forming units (CFU) was counted. In all experimental groups, the number of CFU was minimum in the first cultures, and the results obtained were significantly different from each other at any time period (P < 0.05). In the first culture, the NaOCl group and doxycycline HCl group showed the lowest and highest number of CFU, respectively. In each group, the number of CFU increased significantly by time-lapse (P < 0.05). In conclusion, the substantivity of CHX was significantly greater than NaOCl and doxycycline.