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Chitosan-dextran sulfate nanoparticles of acetazolamide were formulated by using the ionic gelation technique, and evaluated for different attributes like particle size, ζ potential, drug entrapment, particle morphology, in vitro drug release, and in vivo efficacy. Particle size was observed to be changed with the increment of the drug/polymer ratio. Sustained in vitro drug release was exhibited by the particulate formulation that followed the Korsmeyer-Peppas kinetic model. Drug release from nanoparticles was found to occur, as shown by the results, through a combination of dissolution and diffusion. The optimized formulation had a particle size of 172.3 nm and ζ potential of 36.46 mV. The particles had a spherical shape and polydispersity index of 0.257. Decrease in crystallinity of the drug was indicated by powder X-ray diffraction and differential scanning calorimetry studies, in the optimized nanoparticle formulation. Approximately 2.5 times higher transcorneal permeation of drug was observed across the excised goat cornea, in comparison to the aqueous solution of drug, without any corneal damage, during ex vivo transcorneal studies. In vitro mucoadhesion studies showed 91.59% mucoadhesion. The in vivo studies involving ocular hypotensive activity in rabbits revealed significantly higher hypotensive activity with a p-value of