The rationale of current exploration was to formulate positively charged amikacin-loaded polymeric nanoparticles providing a controlled release attribute. Amikacin sulphate-loaded nanoparticles were prepared by w/o/w emulsification solvent evaporation approach succeeded by high-pressure homogenization. Two bioadhesive positively charged polymers, Eudragit Ò RS 100 and Eudragit Ò RL 100, were used in the blend, with variable ratios of drug and polymer. The formulations were assessed in terms of particle size and zeta potential. Thermal gravimetric analysis was brought out on the samples of drug, polymer and drug polymer complex. Drug loading and release attributes of the nanoparticles were scrutinized and antimicrobial activity in contrast to Staphylococcus aureus was appraised. Ocular irritation test, in vivo ocular retention study, in vivo release profile (permeation study) and in vivo antibacterial activity of polymeric nanosuspensions were executed. No rupture consequence but a lengthened drug release was contemplated from all formulations. Amikacin sulphate release from the polymeric nanoparticles reflected a better fit with Korsmeyer-Peppas model. In the course of the antibacterial activity of nanoparticles against S. aureus, formulation AE1 displays the most prominent inhibitory effect as compared with marketed formulation of amikacin sulphate.