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Calcium hydroxide is the “gold standard” endodontic medication, but it may fail to eliminate certain facultative bacteria and yeasts found in root canal systems. In this study, standardized bovine dentin blocks infected with Enterococcus faecalis were treated with an aqueous calcium hydroxide or bioactive glass S53P4 (BAG) powder suspension. While calcium hydroxide was ineffective, the BAG suspension eliminated the infection in the sampled dentin layers after 5 days. In a direct exposure test, preincubation with human dentin boosted the BAG-killing efficacy against E. faecalis ATCC29212, Candida albicans CCUG19915, Pseudomonas aeruginosa ATCC9027, Streptococcus sanguis ATCC10556, and S. mutans ATCC25175. BAG placed in the root canals of extracted teeth did not alter root-dentin pH. Consequently, the conditions under which the microbiota were killed were not pH-mediated. Further studies are ongoing to help clarify the antimicrobial BAG effect in the presence of dentin.