Introduction: 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. Methods: 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 mu g/mL DJK-5, or a mixture of 8.5% EDTA +10 mu g/mL DJK-5 or were combined treated with the solution in the following sequence: 2% or 6% NaOCl +10 mu g/mL DJK-5, 2% or 6% NaOCl + 8.5% EDTA +10 mu g/mL DJK-5, 2% or 6% NaOCl + 8.5% EDTA + 10 mu g/mL DJK-5. Specimens without a smear layer treated by 6% NaOCl or 10 mu g/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. Results: The smear layer reduced the antibacterial capacity of 6% NaOCl and 10 mu g/mL DJK-5. The efficacy of 2% or 6% NaOCl followed by 10 mu g/mL DJK-5 was superior to 10 mu g/ mL DJK-5 alone (P < .05) but inferior to 2% or 6% NaOCl + 8.5% EDTA + 10 mu g/mL DJK-5 and 2% or 6% NaOCl + 8.5% EDTA + 10 mu g/mL DJK-5 (P < .05). The mixture of 8.5% EDTA and 10 mu g/mL DJK-5 had the same disinfection effectiveness as 10 mu g/mL DJK-5 used alone (P < .05). Using 2% or 6% NaOCl before EDTA + peptide always resulted in the highest killing (P < .05). Conclusions: 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.