In this paper, we present the synthesis of nanostructures of magnetite nanoparticles (NPs) with ciprofloxacin and kanamycin antibiotics, based on self-assembling principle. The nanostructures were prepared in crystallite size, ranging 8–16 nm, in one pot addition setup and further washing steps, using only iron precursors and above-mentioned antibiotics as stabilizers. Nanostructures were analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD) analysis methods, Fourier transform infrared (FTIR) and ultraviolet (UV) spectroscopy methods. It was found that they have well-shaped spherical form and are homogeneous in size. The quantitative analysis of nanostructured antibiotics was performed by atom absorbance spectroscopy (AAS) as well as on the basis of Lambert–Beer law. Prepared nanostructures were tested on Staphylococcus aureus and Pseudomonas aeruginosa. Obtained results demonstrated that these nanostructures are able to improve antimicrobial properties and decrease the minimal inhibitory concentration (MIC) of pristine kanamycin and ciprofloxacin antibiotics.