Abstract Introduction Successful endodontic treatment outcomes require new strategies for the complete eradication of microbial biofilms in the root canal system. Matrix metalloproteinases (MMPs) are essential enzymes in microbial cell growth and homeostasis, and they require transition metal ion cofactors to function. Targeting MMP activity also preserves dentin collagen integrity. In this study, 1,10-phenanthroline-5,6-dione (Phendione), a metal chelator, was tested as a potentially novel antimicrobial agent against Enterococcus faecalis and inhibitor of human MMP in the root canal. Methods Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Phendione on E. faecalis were determined. The antimicrobial properties of Phendione in the presence of dentin powder and various transition metal ions were examined. The ability of Phendione to inhibit human MMP-2 was subsequently tested. The efficacy of Phendione against E. faecalis biofilm was determined by exposure of 7-day-old E. faecalis biofilms to Phendione. Results The MIC and MBC of Phendione were 2.0 μg/mL and 16 μg/mL, respectively, whereas 64 μg/mL was required to kill E. faecalis biofilm. Phendione completely eradicated E. faecalis despite dentin preincubation. The presence of Zn2+ , and to a lesser extent Fe2+ , abrogated the antimicrobial effect of Phendione. In addition, Phendione at MIC and MBC significantly inhibited human MMP-2 activity. Conclusions Phendione effectively eradicated E. faecalis biofilms and significantly inhibited human MMP-2 through its ability to chelate metal ions. The antibacterial property of Phendione was preserved in the presence of dentin. Phendione can potentially be applied in endodontic treatment as both an antimicrobial agent and MMP inhibitor.