High intraocular pressure (IOP) is the main risk factor for glaucoma progression. Acetazolamide (AZM) presents a potent IOP-lowering effect but is only administered orally due to its low aqueous solubility and ocular permeability. This study aimed to develop AZM nanocrystals (AZM-NC) as an alternative for its topical ocular delivery. AZM-NC were obtained by wet bead milling technique followed by spray drying, and a mixture design study was conducted to evaluate the optimal drug-to-stabilizer ratio regarding colloidal properties and stability. AZM-NC exhibited an average particle size of 299.73 ± 8.8 nm, a polydispersity index of 0.13 ± 0.01, and a zeta potential of -29.0 ± 0.9 mV, which remained mostly unchanged for at least 60 days when the dried powder was stored at room temperature. Fourier-transformed spectroscopy and powder X-ray diffraction analyses revealed no chemical or crystallinity changes in AZM-NC compared with AZM, respectively. Additionally, AZM-NC demonstrated increased drug saturation concentration, globular shapes, and higher adhesive properties than normal-sized AZM powder. Topical ocular administration of AZM-NC in albino male rabbits showed no clinical signs of ocular damage. Further, in vivo studies revealed a significant IOP reduction of up to 32 % of the basal IOP (-4.8 ± 1.2 mmHg, p <
0.05) in normotensive rabbit eyes (n = 7), after 4 h of AZM-NC suspension topical application, compared to groups treated with AZM suspension, normal saline solution and, AZOPT® (-1.8 ± 1.4 mmHg). Thus, AZM-NC could present a promising approach for developing an eye drop formulation for the localized management of glaucoma.