Biodegradable microparticles containing gentamicin were prepared using chitosan hydroglutamate (CH), hyaluronic acid (HA) and a combination of both polymers by a solvent evaporation method. These formulations were administered nasally via an insufflator. Gentamicin was also administered nasally into rabbits as a solution and powder (a physical mixture of gentamicin and lactose), intravenously (IV) and intramuscularly (IM). The resultant serum levels of gentamicin were determined by Fluorescence Polarisation Immunoassay (FPIA). The bioavailability of gentamicin was poor when administered as a nasal solution (1.1%) and dry powder (2.1%) when compared with IV. However, the microparticulate systems composed of CH and HA/CH considerably enhanced the bioavailability of gentamicin (31.4 and 42.9%, respectively,) with HA microparticles inducing a less significant enhancement (23.3%). Previous in vitro dissolution and frog palate studies indicated that these microparticulate formulations were all mucoadhesive and demonstrated prolonged drug release. Such findings were translated into an increase in the bioavailability of gentamicin when compared with a simple nasal solution in vivo. When HA and CH were combined in the HA/CH formulation, the polymers appeared to improve the absorption of incorporated gentamicin synergistically in comparison to the individual polymers, suggesting a promising nasal delivery system.