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Introduction: This study aimed at assessing the quantitative effect of calcium hydroxide, 2% chlorhexidine gel, and 1.5% chlorhexidine linked to xanthan gel specifically against intratubular bacteria. Methods and Materials: Fifty-two semi-cylindrical bovine dentin specimens were infected with Enterococcus (E.) faecalis by centrifugation with subsequent 7-days incubation. The surface of specimens was disinfected with 3% H202. Scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and the count of bacterial colony-forming units (CFU/mg) were used to assess dentin infection. A total of 40 specimens were incubated for 2 weeks with one of the intracanal medication applied (10 samples for each group): 1) calcium hydroxide, 2) 2% chlorhexidine gel, 3) 1.5% chlorhexidine linked to xanthan gel and 4) sterile saline. Final passive ultrasonic irrigation with 3% sodium hypochlorite was performed in half of the total specimens. The effect of intracanal medications and irrigation against intratubular bacteria was assessed by bacterial culturing of dentin shavings. Two-Way ANOVA model was applied followed by post-hoc Tukey's test for multiple pair-wise comparisons of mean CFU/mg values. Results: SEM, CLSM, and bacterial culturing confirmed the absence of the surface biofilm on the root canal wall and showed vital intratubular bacteria at the depth up to 700 µm. Two-week application of 1.5% chlorhexidine with xanthan gel and 2% chlorhexidine gel significantly decreased intratubular bacterial counts compared with saline (P=0.0003 and P=0.0005, respectively). Subsequent passive ultrasonic irrigation with 3% sodium hypochlorite significantly reduced the number of intratubular bacteria in all groups except for the group with 1.5% chlorhexidine-xanthan gel (P=0.0054). Conclusion: This modified ex vivo model study showed ultrasonically activated irrigation with sodium hypochlorite had greater effect on intratubular bacteria counts compared with 2-week application of intracanal medications. [ABSTRACT FROM AUTHOR]