Since there are more and more cases of multiresistance among microorganisms, rational use of antibiotics (especially their systemic vs. local application) is of great importance. Here we propose polymeric nanoparticles as locally applied gentamicin delivery system useful in osteomyelitis therapy. Gentamicin sulphate (GS) was encapsulated in the poly(lactide-co-glycolide) (PLGA 85:15) nanoparticles by double emulsification (water/oil/water, W1/O/W2). The nanoparticles were characterized by dynamic light scattering, laser electrophoresis and atomic force microscopy. UV-vis spectroscopy (O-phthaldialdehyde assay, OPA) and Kirby-Bauer tests were used to evaluate drug release and antimicrobial activity, respectively. Physicochemical characterization showed that size, shape and drug solubilization of the nanoparticles mainly depended on GS content and concentration of surface stabilizer (polyvinyl alcohol, PVA). Laser electrophoresis demonstrated negative value of zeta potential of the nanoparticles attributed to PLGA carboxyl end group presence. Drug release studies showed initial burst release followed by prolonged 35-day sustained gentamicin delivery. Agar-diffusion tests performed with pathogens causing osteomyelitis (Staphylococcus aureus and Staphylococcus epidermidis, both reference strains and clinical isolates) showed antibacterial activity of GS loaded nanoparticles (GS-NPs). It can be concluded that GS-NPs are a promising form of biomaterials useful in osteomyelitis therapy.