Chelating substances bind to metals, forming stable complexes, rendering these essential ions unavailable for microbial metabolism and community stability in biofilms. This action can contribute to the disinfection in endodontic treatments. Through confocal laser scanning microscopy (CLSM), this study quantified the metal ion removal by chelating agents in Enterococcus faecalis biofilms and assessed the impact on community stability. E. faecalis biofilms were grown for 21 days on acrylic coverslips, which the following were immersed in (n = 10): G1) saline solution (control, 5 min)
G2) 17% ethylenediaminetetraacetic acid (EDTA, 3 min)
and G3) 9% etidronic acid (Dual Rinse HEDP, 5 min). Next, they were stained with Phen Green FL to identify metals, 4',6-diamidino-2-phenylindole (DAPI) to highlight bacterial DNA, and propidium iodide (PI) to mark nucleic acid in cells with damaged membranes. Images were captured (4/sample) by CLSM and analyzed by the software Leica Application Suite X. The individual and total volume of the biofilm-stained components (µm