Background and purpose: Prolonging the drug release can be a suitable approach to overcome the challenges related to topical ophthalmic administration of drugs especially the ones prescribed for chronic ailments. The sustained delivery of the drug would reduce the required frequency of administration which could extremely improve patient compliance and feeling of well-being. This study aimed to develop nanofibrous inserts for sustained ophthalmic delivery of timolol maleate (TIM) for the treatment of glaucoma. Experimental approach: Polycaprolactone-based nanofibers containing TIM were prepared using pure polycaprolactone or a blend of it with cellulose acetate or Eudragit RL100 polymers by the electrospinning method. Following the preparation, polymeric inserts were evaluated for morphological and physicochemical properties. The in vitro drug release was assessed and the in vivo efficacy of a selected insert in decreasing the intraocular pressure (IOP) was also evaluated in the equine eyes. Findings / Results: Prepared nanofibers indicated diameter ranged between 122-174 nm. The formulations showed suitable physicochemical properties and stability for ophthalmic administration. In vitro release study showed prolonged release of drug during more than 3 days. In vivo evaluation revealed that the prepared insert is non-irritant and non-toxic to the equine eyes while having suitable efficacy in decreasing the IOP during 6 days. Conclusions and implication: Prepared TIM inserts indicated a higher efficacy than commercial TIM eye drop in lowering IOP during a prolonged period. Thus, these formulations can be considered suitable for enhancing patient compliance by reducing the frequency of administration in the treatment of glaucoma.