The present report describes the improved ocular retention and aqueous humoral drug availability of ganciclovir (GCV) when administered via topical instillation of different kind of nanoparticles onto the rabbit eye. GCV was loaded into PLGA nanoparticles, chitosan-coated nanoparticles and chitosan-coated niosomal nanoparticles. All three formulations contained nanoparticles equally round in shape with a mean particle size in the range of 180200 nm. The ocular corneal retention property was evaluated by gamma scintigraphy, revealing that the clearance was slowest in the case of the chitosan-containing formulations. GCV in chitosan-coated PLGA nanoparticles and chitosan-coated niosomal nanoparticles showed approx. 6-fold higher aqueous humor drug availability as compared to a GCV solution and nearly 2.5-fold higher as compared to the chitosan-lacking GCV-PLGA nanoparticles. The results indicate that the use of a mucoadhesive chitosan coating can improve the ocular residence time and aqueous humoral availability of GCV when administered topically in nanoparticles.