Conjunctivitis is considered as a common infection of ocular surfaces. Eye drop is most commonly used for treatment of conjunctivitis, but has some drawback like 95% drug eliminated after administration. Administration of levofloxacin to the anterior site in form of chitosan coated poly (lactic-co-glycolic acid) nanoparticles (LFV-CS-PLGA-NPs) expected to overcome these problem and increasing corneal contact time and permeability for effective treatment of bacterial conjunctivitis. The Nanoparticles were developed by single emulsion solvent evaporation technique and optimized for different variables (chitosan, poly (lactide-co-glycolic acid) and polyvinyl alcohol concentration) by employing three factors, three levels Box-Behnken statistical design. The nanoparticles were evaluated for particle size, drug loading, entrapment efficiency, drug release, permeation, ocular tolerance, antimicrobial study, confocal laser microscopy, and Gamma scintigraphy study. The particle size and PdI of the optimized nanoparticles were 169.968 ± 15.23 nm and 0.13 ± 0.03, respectively, where as entrapment efficiency and drug loading is 49.54 ± 2.43% and 11.29 ± 2.13% with extended release profile and strong mucoadhesion. DSC data indicated levofloxacin formed molecular dispersion within coated nanoparticles. Corneal flux showed significantly ( 0.05) higher permeation as compared to marketed formulation. Formulation was nonirritant and possessed good antibacterial activity. Gamma Scintigraphy showed slow drainage compared to drug solution, indicating reduction in nasolachrymal drainage. The Gamma Scintigraphy study indicated the CS coated PLGA-NPs have high corneal residence time as compared to drug solution. So, it is revealed that LFV-CS-PLGA-NPs increase the drug concentration over ocular tissue and potential usefulness for sustained drug delivery.