The aminoglycosidic antibiotic hygromycin B presents a peculiar chemical structure, characterized by two sugar rings joined via a spiro connection. The Cu(ii) complex of hygromycin B in water solution was characterized by (1)H-NMR, UV-Vis, EPR and CD spectroscopy, combined with potentiometric measurements. The spin-lattice relaxation enhancements were interpreted by the Solomon-Bloembergen-Morgan theory, allowing us to calculate copper-proton distances that were used to build a model of the complex by molecular mechanics and dynamics calculations. The fidelity of the proposed molecular model was checked by ROESY maps. Moreover DNA damage by the Cu(ii)-hygromycin B system was also investigated, showing single and double strand scissions exerted by the complex at concentrations in the range 1-5 mM. Addition of either hydrogen peroxide or ascorbic acid to each sample resulted in the shift of the cleavage potency towards lower concentrations of the complex.