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In this study, novel multilayered magnetic nanoparticles (ML-MNPs) loaded with DNase and/or vancomycin (Vanc) were fabricated for eliminating multispecies biofilms. Iron-oxide MNPs (IO-core) (500–800 nm) were synthesized via co-precipitation; further, the IO-core was coated with heavy-metal-based layers (Ag and MoS₂ NPs) using solvent evaporation. DNase and Vanc were loaded onto the outermost layer of the ML-MNP formed by nanoporous MoS₂ NPs through physical deposition and adsorption. The biofilms of S. mutans or E. faecalis (or both) were formed in a brain-heart-infusion broth (BHI) for 3 days, followed by treatment with ML-MNPs for 24 h. The results revealed that coatings of Ag (200 nm) and ultrasmall MoS₂ (20 nm) were assembled as outer layers of ML-MNPs successfully, and they formed Ag-Fe₃O₄@MoS₂ MNPs (3–5 μm). The DNase-Vanc-loaded MNPs caused nanochannels digging and …