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Background: One of the future applications of magnetic nanoparticles is the development of new iron-oxide-based magnetic resonance imaging (MRI) negative contrast agents, which are intended to improve the results of diagnostics and complement existing Gd-based contrast media.Results: Iron oxide nanoparticles designed for use as MRI contrast media are precisely examined by a variety of methods: powder X-ray diffraction (XRD), transmission electron microscopy (TEM), Raman spectroscopy, Mössbauer spectroscopy and zero-field nuclear magnetic resonance (ZF-NMR) spectroscopy. TEM and XRD measurements reveal a spherical shape of the nanoparticles with an average diameter of 5–8 nm and a cubic spinel-type crystal structure of space group Fd−3m. Raman, Mössbauer and NMR spectroscopy clearly indicate the presence of the maghemite γ-Fe2O3 phase. Moreover, a difference in the magnetic behavior of uncoated and human serum albumin coated iron oxide nanoparticles was observed by Mössbauer spectroscopy.Conclusion: This difference in magnetic behavior is explained by the influence of biofunctionalization on the magnetic and electronic properties of the iron oxide nanoparticles. The ZF-NMR spectra analysis allowed us to determine the relative amount of iron located in the core and the surface layer of the nanoparticles. The obtained results are important for understanding the structural and magnetic properties of iron oxide nanoparticles used as T2 contrast agents for MRI.