Glaucoma is characterized by increased intraocular pressure (IOP) that results in blindness if it remains untreated. Acetazolamide (AZM) is a carbonic anhydrase inhibitor, mainly used to reduce IOP in the treatment of glaucoma. However, the potential of topical treatment is limited, due to its low permeability across the ocular epithelium. An alternative to overcome this limitation is the incorporation of AZM in nanoparticulate systems, such as polymeric nanocapsules (NCs). In this way, the aim of this work was to prepare and characterize NC formulations containing AZM, using ethylcellulose (EC) and Eudragit(®) RS100 (EUD) as encapsulating polymers. The formulations showed high encapsulation efficiency. Particle size measurements showed that NCs are in the nanometric range. Comparing both groups of formulations, the NCEC proved to be smaller than those prepared with EUD. The formulations prepared with EC showed negative zeta potentials, while NCs of EUD were positively charged. For both groups of formulations, no more than 30% of drug was released in 120 min. Ex vivo and in vivo studies evidenced that the NCEC formulations were the most efficient, because an increased amount of permeated drug was observed, along with a greater IOP decrease and longer duration of the effect in normotensive rabbits.