Timolol-loaded poly(D,L-lactide-co-glycolide; PLGA) films were prepared for achieving the long-term intraocular pressure (IOP) lowering effect on glaucoma treatment. The physicochemical properties and in vivo effects of films were determined and characterize the delivery system. PLGA, span 20, propylene glycol (PG), and timolol base were dissolved in dichloromethane (DCM) and kept in a stainless mold; timolol films were prepared after the evaporation of DCM. Timolol disc-shape film preparation (TDF), containing 1 mg of timolol in 0.37 cm(2) area, was fabricated by using a trephine and placed onto the cul de sac of alpha-chymotrypsin-induced ocular hypertension rabbits for assessing the IOP lowering effect. The prepared films characterized a Young's modulus ranged from 1.13 to approximately 2.49 MPa, and related to the content percentages of PLGA, PG, and the residual DCM. The timolol film could maintain drug release for 1 week. Following a single-dose application in ocular hypertension rabbits, the prepared TDF could achieve a long-term IOP lowering effect and maintain the IOP change (in comparison with baseline) of approximately 7 mmHg within 1 week. The aqueous humor levels of timolol were low within a range of 0.8 to approximately 0.24 microg/mL for the initial 24 h and less than 0.15 microg/mL for 4-7 days. The investigated film formulation might be potentially developed for the application of long-term ocular delivery systems.