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The objective of this study was to determine the antibacterial efficacy of three calcium hydroxide (CH) formulations using an in vitro model of Enterococcus faecalis dentinal tubule infection. CH mixed with water (CH), CH mixed with iodine-potassium iodide (CH+IKI), and CH mixed with iodoform and silicone oil (Metapex) were tested. Human cylindrical dentin specimens infected with E. faecalis were filled with disinfectants and incubated for 1 week. Dentin powder samples collected with ISO 018 burs showed a statistically significant reduction in E. faecalis for all three experimental groups in comparison with untreated control specimens. Statistically significant differences were also found between the three experimental groups. Metapex was the most effective dentinal tubule disinfectant, followed by CH+IKI and then CH. Similar results were observed at greater dentin tubule depths (ISO 021 burs) with the exception that intracanal treatment with CH resulted in significantly higher numbers of E. faecalis in comparison with untreated control specimens.