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The present study aimed to evaluate the antibacterial effect of a new peptide, DJK-5, used alone or mixed together with EDTA on mono- and multispecies biofilms in dentin canals covered by a smear layer with or without preceding sodium hypochlorite (NaOCl) irrigation.One hundred twelve dentin blocks (224 final specimens) were prepared and divided into 56 groups, and Enterococcus faecalis or multispecies bacteria were introduced into dentinal tubules by centrifugation. After 1 week of cultivation, a uniform smear layer was created on the surface of the dentin blocks, and the samples were exposed to sterile water, 17% EDTA, 2% or 6% NaOCl, 10 μg/mL DJK-5, or a mixture of 8.5% EDTA +10 μg/mL DJK-5 or were combined treated with the solution in the following sequence: 2% or 6% NaOCl +10 μg/mL DJK-5, 2% or 6% NaOCl + 8.5% EDTA +10 μg/mL DJK-5, 2% or 6% NaOCl + 8.5% EDTA + 10 μg/mL DJK-5. Specimens without a smear layer treated by 6% NaOCl or 10 μg/mL DJK-5 served as the positive control. The irrigant exposure time was 3 or 10 minutes. The antibacterial efficacy was determined by live/dead staining and confocal laser scanning microscopy.The smear layer reduced the antibacterial capacity of 6% NaOCl and 10 μg/mL DJK-5. The efficacy of 2% or 6% NaOCl followed by 10 μg/mL DJK-5 was superior to 10 μg/mL DJK-5 alone (P < .05) but inferior to 2% or 6% NaOCl + 8.5% EDTA + 10 μg/mL DJK-5 and 2% or 6% NaOCl + 8.5% EDTA + 10 μg/mL DJK-5 (P < .05). The mixture of 8.5% EDTA and 10 μg/mL DJK-5 had the same disinfection effectiveness as 10 μg/mL DJK-5 used alone (P < .05). Using 2% or 6% NaOCl before EDTA + peptide always resulted in the highest killing (P < .05).The smear layer inhibits the disinfectant effect in dentin. Peptide DJK-5 showed a strong antibacterial effect against mono- and multispecies biofilms in dentin canals. The highest killing was measured when 6% NaOCl was followed by a mixture of EDTA and peptide DJK-5.