OBJECTIVES:  In this study, we aimed to evaluate the bactericidal effect and cytotoxicity of an ethylenediaminetetra-acetic acid (EDTA)-based root canal irrigant solution capable of efficiently removing both the organic matter and the smear layer. We prepared a strong alkaline EDTA (AE) solution with an acid buffer capacity similar to that of sodium hypochlorite. MATERIALS AND METHODS:  AE was used at concentrations of 1%, 2%, and 3%. The bactericidal effect of AE on Enterococcus faecalis was evaluated by determining the colony number and biofilm removal rate. Biofilms were visualized using a Live/Dead BacLight bacterial viability kit. Viability of AE-treated cells were determined using a CCK-8 cell counting assay. STATISTICAL ANALYSIS:  One-way analysis of variance followed by a Dunnett's multiple comparison test were used for comparisons among groups. RESULTS:  Significant reduction in cell viability and biofilm formation were observed in case of 3% and 2% AE. AE exerted bactericidal effects in a concentration-dependent manner. Damage of normal human fibroblasts was not observed at any of the AE concentrations. CONCLUSIONS:  Our results suggest that the AE solution could be used as an effective canal irrigant for the removal of bacterial biofilm.