Polyamidoamine dendrimers have been investigated as a potential platform for a number of ocular drugs but only in aqueous solution. In this work, we have developed fast dissolving dendrimer-based nanofibers (DNF) as a topical delivery vehicle for the glaucoma drug brimonidine tartrate (BT). The safety and drug release kinetics of these nanofiber mats were evaluated in vitro and in vivo. DNF caused no toxicity at therapeutic levels in cultured cells or ocular irritation in animal tests using a normotensive rat model. Intraocular pressure response was equivalent between DNF and BT solutions in a single dose test, but DNF showed significantly improved efficacy with daily dosing over a 3-week test period. This study indicates electrospun dendrimer nanofibers are a viable alternative to eye drops as a more efficient method of topically administering antiglaucoma drug.