Purpose: The aim of this study was to prepare a novel oil-in-water microemulsion of ofloxacin (OFX) for topical ocular application. Methods: Pseudo-ternary phase diagrams were constructed for combination of oleic acid as oil phase, Tween 80 as surfactant, ethanol as co-surfactant, and 0.5N NaOH solutions as aqueous phase. The optimum microemulsion was modified with 0.75% chitosan oligosaccharide lactate (COL). The properties of microemulsions in the absence or presence of OFX (0.3%) were measured, such as electrical conductivity, droplet size, viscosity, and pH. The in vitro release study was carried out using the dialysis bag method. Ex vivo permeability studies were performed with rabbit cornea in Franz-diffusion cells. Sterility, minimum inhibition concentration (MIC), and antibacterial activity studies were conducted microbiologically. The preocular residence time and efficacy against bacterial keratitis was compared with a commercial (C) solution via in vivo studies. Results: M2OFX modified with 0.75% COL showed slower release than M1OFX, which does not contain COL. The permeation rate of OFX from M1OFX was significantly higher than M2OFX and the C solution. The formulations were sterile and MIC values were the same for both. M2OFX, which contains 0.75% COL, performed higher antibacterial activity than M1OFX. The preocular residence time was improved by microemulsion in comparison to solution; the addition of COL did not make a significant difference. In total, 8 rabbits gave better results with M1OFX, whereas 4 gave similar scores to commercial solution-applied rabbits. Conclusion: In conclusion, OFX microemulsions could be offered as a promising strategy for ocular drug delivery.