Objectives: The aim of the present study was to develop novel ofloxacin (OFX)-loaded nanostructured lipid carrier (NLC)-based inserts for ocular application for treatment of bacterial keratitis.Methods: NLC loaded with 0.3% OFX was prepared by means of high shear homogenization and 0.75% chitosan oligosaccharide lactate (COL) was added. Glycerin or PEG 400 at the range of 1 - 15% was added to NLCs as plasticizers and inserts were developed by solvent casting evaporation. Characterization, in vitro release, microbiological, ex vivo and in vivo studies were performed.Results: The inserts developed with the addition of glycerin (Ins3(OFX)) was found as optimal. The kinetic studies revealed that the release of Ins3(OFX) was a combination of diffusion and swelling. Ins3(OFX) was more bioadhesive in texture profile analysis studies. In the in vivo studies performed with rabbits, the pre-ocular retention time was enhanced up to 24 h and C-max was increased almost six times in comparison with commercial. The rabbits were infected with Staphylococcus aureus and keratitis was confirmed. This group was treated with Ins3(OFX) and they recovered in 7 days with no significant sign of conjunctival redness and corneal opacity.Conclusion: NLC-based inserts developed with COL and glycerin may be offered as appropriate vehicles for ocular delivery.