Chemical stability and good biocompatibility under physiological conditions render nanoparticles made of the spinel oxide magnetite a favorable choice in biomedical diagnostic and therapeutic applications that benefit from high levels of magnetization and superparamagnetism. Under ambient atmosphere, however, magnetite nanoparticles are prone to undesired oxidation leading to an at least partially oxidized form of magnetite. In the present work Fe3-xO4 nanopowders (with a particle size of 10-50 nm) were prepared under different conditions via chemical co-precipitation method, resulting in samples with different oxidation levels. The effect of oxidation of the prepared samples on their morphological, structural, electronic and magnetic properties is followed, respectively, by the means of transmission electron microscopy (TEM), powder X-ray diffractometry (PXRD),Fe-57 Mossbauer spectroscopy (MS) and electron magnetic resonance (EMR) spectroscopy measurements. A novel method is applied to decompose the heavily broadened room-temperature Fe-57 Mossbauer spectra of the nanoparticles into signals of intermediate valence Fe2.5+ and that of Fe3+ iron species. The results indicate that the cubic lattice parameter of non-stoichiometric magnetite nanoparticles depends on the concentration as well as on the mean oxidation level of the intermediate valence iron species. At the same time, EMR spectra of the samples indicate that oxidation influences the magnetic anisotropy of the nanoparticles, with the magnitude of the nanoparticles' magnetic anisotropy field being correlated with the concentration of the intermediate valence iron species. Malic acid, used as coating agent for several of the samples, is shown to hinder the oxidation of magnetite nanoparticles.