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In this study, we prepared chitosan/Fe(III)/deferoxamine nanoparticles with unimodal size distribution (hydrodynamic diameter ca. 250 nm, zeta potential ca. 32 mV). The elaborated nanoparticles are characterized by outstanding in vitro and in vivo antibacterial activity, which exceeds even that of commercial antibiotics ampicillin and gentamicin. Moreover, the nanoparticles are non-toxic. We found that the introduction of iron ions into the chitosan matrix increases the ability of the resulting nanoparticles to disrupt the integrity of the membranes of microorganisms in comparison with pure chitosan. The introduction of deferoxamine into the obtained nanoparticles sharply expands their effect of destruction the bacterial membrane. The obtained antibacterial nanoparticles are promising for further preclinical studies.