A new water-dispersible nanostructure based on magnetite (Fe3O4) and usnic acid (UA) was prepared in a well-shaped spherical form by a precipitation method. Nanoparticles were well individualized and homogeneous in size. The presence of Fe3O4@UA was confirmed by transmission electron microscopy, Fourier transform-infrared spectroscopy, and X-ray diffraction. The UA was entrapped in the magnetic nanoparticles during preparation and the amount of entrapped UA was estimated by thermogravimetric analysis. Fabricated nanostructures were tested on planktonic cells growth (minimal inhibitory concentration assay) and biofilm development on Gram-positive Staphylococcusaureus (S.aureus),Enterococcus faecalis (E.faecalis) and Gram-negative Escherichia coli (E.coli),Pseudomonasaeruginosa (P.aeruginosa) reference strains. Concerning the influence of Fe3O4@UA on the planktonic bacterial cells, the functionalized magnetic nanoparticles exhibited a significantly improved antimicrobial activity against E.faecalis and E.coli, as compared with the Fe3O4 control. The UA incorporated into the magnetic nanoparticles exhibited a very significant inhibitory effect on the biofilm formed by the S.aureus and E.faecalis, on a wide range of concentrations, while in case of the Gram-negative microbial strains, the UA-loaded nanoparticles inhibited the E.coli biofilm development, only at high concentrations, while for P.aeruginosa biofilms, no inhibitory effect was observed. The obtained results demonstrate that the new water-dispersible Fe3O4@UA nanosystem, combining the advantages of the intrinsic antimicrobial features of the UA with the higher surface to volume ratio provided by the magnetic nanocarrier dispersible in water, exhibits efficient antimicrobial activity against planktonic and adherent cells, especially on Gram-positive strains.