Oral biofilm-related infections remain a persistent and costly clinical problem. Existing treatments are unable to simultaneously kill and physically disrupt biofilms and require manual biofilm removal procedures that are cumbersome with limited efficacy in difficult to reach areas such as the endodontic canal systems. New efficacious, automated technologies capable of precisely targeting complex anatomical areas are needed to kill bacteria as well as degrade and remove biofilm structure. We propose a novel approach that combines nanotechnology and robotics to develop the first automated biofilm eradication platform. We have designed small-scale robots using catalytic nanoparticles as building blocks that display tether-free controlled motion with multifunctionality. Our approach exploits iron oxide nanoparticles (IONPs) with dual catalytic-magnetic properties that (i) generate bactericidal and biofilm degrading reactive molecules in situ, and (ii) remove the disrupted biofilm via magnetic-field driven robotic assemblies termed Catalytic Antibiofilm Robots (CARs). Preliminary data demonstrate that CARs locally target and remove biofilms with high precision and efficacy, including confined endodontic spaces. We hypothesize that, by tuning CARs magneto-catalytic properties to enhance the ‘kill-degrade-and-remove’ mechanism, improved antibiofilm efficacy and maneuverability can be achieved for targeted endodontic disinfection and drug delivery. To achieve this, we propose (Aim 1) to improve the catalytic and magnetic properties of IONP building blocks via physicochemical modifications including particle size and surface functionalization to enhance catalysis, biofilm targeting and controllability. We will assess catalytic efficiency, magnetic field response, and bioactivity to identify key parameters for CAR improvement. Then, optimized IONPs will be incorporated into two CARs platforms. CAR1s, formed from aggregated IONP, will be used for catalytic bacterial killing and biofilm treatment in root canal systems (Aim 2). We will determine the principles governing magnetic field response and antibiofilm activity, and test the efficacy of these robots to remove biofilms in difficult-to-reach areas in a controlled manner. We will assess bioactivity and maneuverability considering key anatomical and biological complexities using mixed-species biofilm and diverse canal morphologies recapitulated in 3D-printed teeth and ex vivo models. In the second platform, CAR2s will be fabricated by 3D micromolding of functional polymers with embedded IONPs for biofilm removal and drug delivery in the interior of tooth canal (Aim 3). We will study and optimize magnetic control, antibiofilm activity and triggered cargo delivery conditions. Thereafter, we will apply information from the mechanistic studies to design and evaluate CAR2s for simultaneous biofilm removal and drug delivery at the apical region using 3D printed and ex vivo tooth models with mixed-species biofil...