The aim of this in vitro investigation was to use a recently developed Enterococcus faecalis infection model using root canal for evaluating iron oxide (Fe3O4) nanoparticles (NP) with biomimetic (catalytic) properties as a new antimicrobial endodontic treatment. We compared iron oxide NP bioactivity with currently used chemical modalities using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) as analytical tools. We hypothesized that iron oxide NP with enzyme-like (peroxidase) activity catalyzes H2O2 to promote bacterial killing within dentinal tubules (DT) via in situ production of free radicals. We further hypothesized that the NP is more effective than the conventional treatments (irrigants) used in the clinical endodontic practice. Because iron oxides can be used as food additives, and iron oxide NP formulations are low-cost and FDA-approved for human use, it could be a safe and feasible approach to potentiate the effects of a commonly used antiseptic.