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The purpose of this study was to determine the influence of dentin powder on the concentration, pH, and antimicrobial activity of sodium hypochlorite (NaOCl) alone and combined with etidronic acid (HEBP).Biofilms of Enterococcus faecalis were grown on the surface of dentin blocks for 5 days and then exposed to 1% and 2.5% NaOCl alone or combined with 9% HEBP for 3 minutes in the absence and presence of dentin powder. The biovolumes of the biofilm were measured using confocal microscopy and the live/dead technique. The available chlorine and pH of the solutions were also measured. Nonparametric tests were used to determine statistical differences (P < .05).The presence of dentin powder resulted in a reduction of the free available chlorine and pH in all the irrigating solutions; 1% NaOCl lost its antimicrobial activity completely in the presence of dentin powder. The antimicrobial activity was significantly reduced in the 2.5% NaOCl and 1% NaOCl/HEBP groups, and it was not affected in the 2.5% NaOCl/HEBP group.The presence of dentin powder significantly decreased the available chlorine and antimicrobial activity of 1% NaOCl, 2.5% NaOCl, and 1% NaOCl/HEBP irrigating solutions. The antimicrobial activity of 2.5% NaOCl/HEBP was not affected by the dentin powder after a 3-minute contact time against E. faecalis biofilms.Dentin powder decreases the activity of 1% sodium hypochlorite (NaOCl), 2.5% NaOCl, and 1% NaOCl/etidronic acid (HEBP).The antimicrobial activity of 2.5% NaOCl/HEBP is not affected by dentin powder.HEBP does not reduce the antimicrobial properties of NaOCl when combined.