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Numerous studies have reported the possibility of enhancing the properties of materials by incorporating foreign elements within their crystal lattice. In this context, while magnetite has widely known properties that have been used for various biomedical applications, the introduction of other metals within its structure could prospectively enhance its effectiveness. Specifically, zinc and cerium have demonstrated their biomedical potential through significant antioxidant, anticancer, and antimicrobial features. Therefore, the aim of the present study was to develop a series of zinc and/or cerium-substituted magnetite nanoparticles that could further be used in the medical sector. The nanostructures were synthesized through the co-precipitation method and their morpho-structural characteristics were evaluated through X-ray diffraction (XRD), inductively coupled plasma mass spectrometry (ICP-MS), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), zeta potential, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) analyses. Furthermore, the nanostructures were subjected to a ROS-Glo H2O2 assay for assessing their antioxidant potential, MTT assay for determining their anticancer effects, and antimicrobial testing against S. aureus, P. aeruginosa, and C. albicans strains. Results have proven promising for future biomedical applications, as the nanostructures inhibit oxidative stress in normal cells, with between two- and three-fold reduction and cell proliferation in tumor cells; a two-fold decrease in cell viability and microbial growth; an inhibition zone diameter of 4–6 mm and minimum inhibitory concentration (MIC) of 1–2 mg/mL. [ABSTRACT FROM AUTHOR]