INTRODUCTION: The aim of this study was to evaluate the effect of DJK-5, a newly developed cationic antimicrobial peptide, on oral multispecies and Enterococcus faecalis biofilms alone or combined with the endodontic chelating agent EDTA in vitro. METHODS: Oral multispecies biofilms from 2 donors and E. faecalis VP3-181 biofilm were grown on collagen-coated hydroxyapatite disks. After incubation for 3 days or 3 weeks, the biofilms were exposed to sterile saline (negative control), 8.5% EDTA, 2% chlorhexidine digluconate (CHX), 5 μg/mL DJK-5, 10 μg/mL DJK-5, a mixture of 5 μg/mL DJK-5 and 8.5% EDTA (final concentration), or a mixture of 10 μg/mL DJK-5 and 8.5% EDTA, all for 1 and 3 minutes. The proportions of dead bacteria in the biofilms were assessed by the LIVE/DEAD staining (Thermo Fisher Scientific, Waltham, MA) and confocal microscopy. RESULTS: The peptide DJK-5 rapidly killed most bacteria in all biofilms, with significant differences to the control, 8.5% EDTA and 2% CHX (P < .01). Basically, a higher DJK-5 concentration and longer exposure (3 minutes) were more effective than a low concentration and short time exposure (P < .05). There were no significant differences in antibiofilm activities between DJK-5 used alone or in the mixture with 8.5% EDTA at either concentration. EDTA (8.5%) had no significant antimicrobial effect compared with the negative control (P > .05), but, unlike DJK-5 alone, the mixture retained the ability to remove smear layers. In peptide groups, there were no significant differences in dead bacteria proportions between 3-day and 3-week biofilms, except for 10 μg/mL DJK-5 used alone for 3 minutes on the multispecies biofilms. CONCLUSIONS: DJK-5 exerted antibiofilm ability on E. faecalis and oral multispecies biofilms grown on hydroxyapatite disks, both alone and when combined with 8.5% EDTA.