Ferumoxytol (Fer) is an Iron oxide nanoparticle formulation (IONP) that have been FDA-approved for systemic administration to treat iron deficiency anemia. Several IONP formulations have been approved for different biomedical applications including MRI, drug delivery and anti-inflammatory treatment. Moreover, IONPs exhibit strong catalytic activity that mimic natural horseradish peroxidase enzyme, which has shown therapeutic activity including prevention of tumors and infections. Many infectious diseases are caused by and associated with biofilms, including endodontic infections. The reduction or elimination of microorganisms has been associated with positive endodontic outcomes and is the basis for prevention and treatment of endodontic disease. However, conventional antimicrobial agents fail to completely eliminate bacterial biofilm within the root canal system. Here, we show that ferumoxytol nanoparticles (an FDA-approved formulation of iron oxide nanoparticles) are a potent antibiofilm agent and an effective endodontic irrigation solution for disinfection of the root canal system. Ferumoxytol (Fer) displays catalytic activity similar to peroxidase at acidic pH to activate hydrogen peroxide (H2O2) to release antimicrobial free v radicals to eradicate endodontic biofilms. Further analyses revealed that Fer nanoparticles can bind to biofilm and maintain their catalytic activity in situ. Notably, Fer/H2O2 topical therapy demonstrated enhanced antimicrobial activity in comparison to a standard of care solution (NaOCl) against ex vivo mixed-species biofilm. Furthermore, Fer/H2O2 was effective against endodontic infections in patients diagnosed with pulp necrosis and apical periodontitis with no reported adverse effects. Intriguingly, Fer nanoparticles were devoid of cytotoxic effects on stem cells of the apical papilla (SCAPs). Altogether, Fer nanoparticles are a potent and biocompatible antibiofilm agent in both laboratory and human studies, demonstrating promising therapeutic potential as a novel disinfection approach. Our data show a promising yet translatable therapeutic platform based on iron oxide nanoparticles that could have immediate clinical applications to combat endodontic infections and other oral biofilm disease.