This study describes corneal permeation of gatifloxacin from solid lipid nanoparticle (SLN) through the cornea and its effect on corneal hydration level. The aqueous humor levels of gatifloxacin after single topical instillation in Gate(®) Eyedrops and positively charged SLN-C were determined. A 3.37-fold increase in the relative bioavailability was observed with the SLN-C (AUC0→∞ 2.192 μg mL(-1) h) (AUC, area under the curve) as compared to Gate(®) Eyedrops (AUC0→∞ 0.651 μg mL(-1) h). The t1/2 of drug in SLN-C exhibited a 2.34-fold higher than Gate(®) Eyedrops seem to be significantly increased (p > 0.05), Cmax of gatifloxacin from SLN-C showed 1.09-fold higher concentration as compared to Gate(®) Eyedrops. The results suggested that SLNs could enhance ocular bioavailability of gatifloxacin and prolong its residence time in the eyes. Moreover, no signs of ocular irritation were seen with the SLN formulations, indicating their relative safety compared to the marketed drops.