Statement of problem: The main purpose of root canal treatment is to eliminate microorganisms and their by-products. The use of antimicrobial agents besides mechanical preparation is necessary to achieve this goal. On the other hand, calcium hydroxide is ineffective in Killing some resistant microorganisms. Purpose: The aim of this in vitro study was to compare the effectiveness of calcium hydroxide mixed with different vehicles (Distilled water, Sodium hypochlorite or Chlorhexidine) in disinfecting the root canal and dentin of the infected teeth.Materials and Method: After root canal preparation of 130 single rooted teeth, they were sterilized and randomly divided into three experimental (n= 40 in each group) and two control groups. Then, the specimens were experimentally infected with E. faecalis and C. albicans during a period of 21 days. The infected specimens were exposed to the pastes of calcium hydroxide mixed with distilled water solution, CHX or NaOCl. Antimicrobial assessment was performed at 24h, 48h, 1 week and 2 week (n=10 in each time interval). Microbial samples were obtained from the root canals before and after the experiment. Also, the dentin samples were obtained for evaluating dentin disinfection. The data were analyzed, using Two-way ANOVA test.Results: This study showed that there was no significant difference between the three intracanal medicaments and also between the four periods of time ( p >0.05). However, the mixture of calcium hydroxide/distilled water did not completely eradicate E. faecalis from dentinal tubules at any period of time.Conclusion: According to the results of this study, calcium hydroxide mixed with CHX or NaOCl is better than calcium hydroxide mixed with distilled water for dentin disinfection in cases of persistent intracanal infection.Key words: Enterococcus faecalis, Calcium hydroxide, Candida albicans, Chlorhexidine, Root canal medicaments, Sodium hypochlorite