The current research focuses on developing positively charged gelatin nanoparticles loaded with moxifloxacin for its effective ocular delivery and controlled release in corneal eye layer. We selected type A gelatin because of its biodegradable and non-toxic nature as the polymer of choice for fabricating the nanoparticles by a modified two step desolvation technique. The produced nanoparticles were positively charged (+24 +/- 0.12 mV) with a narrow particle size of 175 +/- 1.11 nm as measured by dynamic light scattering (DLS). The in-vitro drug release from the nanoformulations exhibited a burst effect in the first hour followed by a controlled release of the drug for the subsequent 12 h. The Korsmeyer-Peppas model showed better linearity and the formulations displayed non-Fickian drug release pattern. The optimized formulation was assessed for its utility as an anti-bacterial agent and its effectiveness was tested on the corneal eye surface of rabbits. The in-vivo tolerance tests revealed that the drug loaded nano formulations was non-irritant to the ocular tissues indicating its safety. The in-vivo anti-bacterial activity of the nanosuspension was more effective against S. aureus than the commercially market product, MoxiGram (R). Microbiological efficacy assessed against B. subtilus using cup-plate method suggested that our fabricated nanosuspension possess better anti-microbial activity as compared to the commercial agent, MoxiGram (R) revealing promising potentials for the currently developed gelatin based nanoformualtions. (C) 2016 Elsevier Inc. All rights reserved.