The persistence of anaerobic bacteria in the root canal system often leads to treatment failure. One possible reason for this may be the retention of micro-organisms in the dentinal tubules of root canal walls. This study was performed to compare the effectiveness of two root canal medicaments and a chlorhexidine solution in disinfecting Actinomyces israelii-infected root canal walls and dentinal tubules in vitro. Dentinal tubules of root canal walls of human teeth were experimentally infected with A. israelii. The root canals were exposed to either iodine potassium iodide, calcium hydroxide or 2% chlorhexidine for periods of 3, 7 and 60 days. At the end of the medication periods samples were removed at different depths and tested for A. israelii viability. Chlorhexidine was the only disinfectant that was able to eliminate A. israelii from all the samples after 3, 7 as well as 60 days while 25% of the specimens treated with iodine potassium iodide and 50% of the specimens treated with calcium hydroxide still had viable A. israelii after treatment. It is clear from this study that 2% chlorhexidine is superior to iodine potassium iodide and calcium hydroxide in its ability to remove A. israelii from infected dentinal tubules. However, in vivo trials need to be undertaken before its clinical use can be recommended.