Amphotericin B (AmB) has a broad antifungal and leishmanicidal activity with low incidence of clinical resistance. Its parenteral administration has high risk of nephrotoxiciy that limits its use. In order to treat cutaneous infections, AmB topical administration is a safer therapy because of the low systemic absorption of the drug across mucous membranes. Moreover, in some developing countries both fungal topical infections and cutaneous leishmaniosis are an important health problem. The aim of this work is to formulate a topical amphotericin preparation and test its in vitro antifungal (against 11 different fungal species) and antileishmanial activity. γ-Cyclodextrin (γ-CD) was chosen to solubilise AmB. Furthermore, γ-CD has shown a synergistic effect on membrane destabilization with AmB. Topical novel formulations based on AmB-CD complex have exhibited greater antifungal activity (48%, 28% and 60% higher) when compared to AmB Neo-Sensitabs^® disks, AmB dissolved in dimethyl sulfoxide (DMSO) and Clotrimazole^® cream, respectively. Furthermore, AmB-CD methyl cellulose gel has shown significantly higher inhibition activity on biofilm formation, larger penetration through yeast biofilms and higher fungicidal activity on biofilm cells compared to AmB dissolved in DMSO. In addition, AmB-CD gel exhibited both high in vitro leishmanicidal efficacy with wider therapeutic index (between 2 and 8-fold higher than AmB deoxycholate depending on Leishmania spp.) and also in vivo activity in an experimental model of cutaneous leishmaniasis. These results illustrate the feasibility of a topical AmB formulation easy to prepare, physicochemically stable over 6 months, safe and effective against diverse fungal and parasitic cutaneous infections. © 2014 Elsevier B.V.