The aim of this study was to evaluate the anti-biofilm and anti-metabolic activities of zeolite-zinc oxide nanoparticles (Zeo/ZnONPs)-based antimicrobial photodynamic therapy (aPDT) against pre-formed polymicrobial biofilms on the orthodontic brackets, as well as, assess the remineralization efficacy on polymicrobial biofilms induced enamel lesions. Following synthesis and characterization of Zeo/ZnONPs, cell cytotoxicity, hemolytic effect, and intracellular reactive oxygen species (ROS) production were determined. The anti-biofilm and anti-metabolic activities of aPDT using different concentrations of Zeo/ZnONPs were investigated. Microhardness tester and DIAGNOdent Pen were used to evaluate the changes of remineralization degree on the treated enamel slabs duration 1 and 3 months. No significant cytotoxicity and erythrocyte hemolysis were observed in treated cells with Zeo/ZnONPs. When irradiated, suggesting that the Zeo/ZnONPs were photoactivated, generating ROS and leading to reduce dose-dependently the cell viability and metabolic activity of polymicrobial biofilms. Also, the enamel surface microhardness value of exposed enamel showed a steady increase with the concentration of Zeo/ZnONPs. No statistically significant differences were shown between aPDT and sodium fluoride varnish as the control group. Overall, Zeo/ZnONPs-based aPDT with the greatest remineralization efficacy of enamel surface can be used as an anti-biofilm therapeutic method, which is involved with their potent ability to produce ROS.