To compare the anti-biofilm efficacy of two antimicrobial peptides (AMPs), 1018 and DJK-5, in disrupting canal wall biofilms in the isthmus, canal and dentinal tubules of single-rooted maxillary premolars.,Enterococcus faecalis single-species biofilms were formed in-situ in the root canal system of the premolars (n = 91). Confocal laser scanning microscopy, bacterial sampling, colony-forming unit counting, XTT assay, lactate dehydrogenase assay and phenol-sulphuric acid method were used to identify the anti-biofilm efficacy of both AMPs and their influence on bacterial metabolic activity.,Both AMPs disrupted in-situ E. faecalis biofilms and altered their metabolic activity. At 20 μg/mL, the d-enantiomeric AMP DJK-5 killed 55.5 %, 57.3 % and 55.8 % of biofilm bacteria in the isthmus, canal and dentinal tubules, respectively, in 1 min. In contrast, the l-enantiomeric AMP 1018 only eradicated 25.6 %, 25.5 % and 27.5 % of biofilm bacteria in the isthmus, canal and dentinal tubules, respectively, within the same time. Anti-biofilm efficacy of the root canal irrigants tested were in the order: 6 % NaOCl > 20 μg/mL DJK-5 > 10 μg/mL DJK-5 > 20 μg/mL 1018 > 10 μg/mL 1018 > 0.9 % NaCl.,The present results are confirmatory of previous studies, in that d-enantiomeric AMPs exhibit more potent antibacterial properties than l-enantiomeric AMPs against E. faecalis biofilms within the canal space. Nevertheless, the potency of both AMPs are concentration-dependent. Incorporation of these agents into EDTA, a non-antibacterial calcium-chelating irrigant for removal of the inorganic component of the canal space debris, does not reduce the efficacy of either AMP.,The present study provides the proof of concept that incorporation of an antimicrobial peptide into a calcium-chelating root canal irrigant enhances the disinfection of intratubular single-species biofilms during smear layer and smear plug removal.,Published by Elsevier Ltd.