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The emergence of antibiotic‐resistant bacterial infections is a principal threat to global health. Functionalization of nanomaterial with antibiotics is known as a useful method for increasing the effectiveness of existing antibiotics. In this study, vancomycin‐functionalized ZnFe2O4 nanocomposite (ZnFe2O4@Cell@APTES@Van) was synthesized, and its functional groups and particle size were characterized using Fourier‐transform infrared spectroscopy, thermogravimetric analysis, dynamic light scattering, scanning electron microscope, and transmission electron microscopy. The antibacteria activity of the synthesized nanocomposite was evaluated using minimum inhibitory concentration and minimum bactericidal concentration against Escherichia coli, Staphylococcus aureus, and methicillin‐resistant Staphylococcus aureus (MRSA). Cytotoxicity assay was done by 2,3‐bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide method. Characterization analyses of synthesized nanocomposite confirmed the binding of vancomysin on the surface of ZnFe2O4@Cell@APTES. Nanocomposite exhibited an aggregated semi‐spherical structure with hydrodynamic radii of ∼382 nm. In vitro antibacterial activity test showed that vancomycin and vancomycin functionalized ZnFe2O4 have no antibacterial effect against E. coli. S. aureus was sensitive to vancomycin and ZnFe2O4@Cell@APTES@Van NPs and ZnFe2O4 NPs did not improve vancomycin antibacterial activity against these bacteria. MRSA is resistant to vancomycin while ZnFe2O4@Cell@APTES@Van NPs was efficient in inhibiting MRSA growth. In summary, this study showed that attachment of vancomycin to ZnFe2O4 NPs was increased its antibacterial activity against MRSA. [ABSTRACT FROM AUTHOR]