Amphotericin B (AmB) is a wide spectrum antifungal with low incidence of clinical resistance. However, there are no licensed topical formulations with AmB in most developed countries. Extemporaneous preparations of AmB are frequently prepared from available marketed parenteral formulations. Herein, a solution of AmB with γ-cyclodextrin is described as suitable for topical administration as eye drops. This novel formulation is characterised by its ability to solubilise AmB and to maintain its antifungal activity, physicochemical stability and sterility over 30 days. Antifungal activity against Candida albicans was significantly higher (35%) for the new formulation than that obtained with Fungizone® based extemporaneous prepared suspension. Optimal 0.1% AmB-10% cyclodextrin formulation remained sterile and with an acceptable osmolarity, pH and particle size for ophthalmic use over 4 weeks. Complexation with γ-cyclodextrins improved AmB chemical stability compared to the reference eye drops suspension based on Fungizone®. These results illustrate the feasibility of an ophthalmic AmB formulation easy enough to be licensed or prepared in community and hospital pharmacies. © 2012 Elsevier B.V. All rights reserved.