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Thorough disinfection of the root canal system is essential for the success of root canal therapy. This requires the use of an intra-canal medicament. Enterococcus faecalis is the most frequently found species in persistent/secondary intracanal infection associated endodontic treatment failure. This study evaluates the disinfection of dentinal tubules using 2% chlorhexidine gel, 2% metronidazole gel, bioactive glass (S53P4) in comparison with calcium hydroxide. The antibacterial efficacy of the four medicaments against Enterococcus faecalis was assessed in vitro using extracted premolar teeth at the depths of 200 μm and 400 μm. The overall percentage inhibition of bacterial growth (at 200 μm and 400 μm depth) was 100% with 2% chlorhexidine gel. The inhibition of growth was moderate with 2% metronidazole gel (86.5%), followed by bioactive glass (62.8%) and calcium hydroxide (58.5%). It can be concluded …