Aiming to improve the topical delivery of AmB to treat cutaneous fungal infections and leishmaniasis, ultradeformable liposomes containing amphotericin B (AmB-UDL) were prepared, and structural and functional characterized. The effect of different edge activators, phospholipid and AmB concentration, and phospholipid to edge activator ratio on liposomal deformability, as well as on AmB liposomal content, was tested. Liposomes having Tween 80 as edge activator resulted of maximal deformability and AmB/phospholipid ratio. These consisted of AmB-UDL of 107±8nm diameter, 0.078-polydispersity index and -3±0.2mV Z potential, exhibiting monomeric AmB encapsulated in the bilayer at a 75% encapsulation efficiency. After its cytotoxicity on keratinocytes (HaCaT cells) and macrophages (J774 cells) was determined, the in vitro antifungal activity of AmB-UDL was assayed. It was found that fungal strains (albicans and non-albicans Candida ATCC strains and clinical isolates of C. albicans) were more sensitive to AmB-UDL than mammal cells. Minimum inhibitory concentration values for AmB-UDL were 5-24 and 24-50 times lower than IC50 for J774 and HaCaT cells, respectively. AmB-UDL at 1.25μg/ml also displayed 100 and 75% anti- Leishmania braziliensis promastigote and amastigote activity, respectively. Finally, upon 1h of non-occlusive incubation, the total accumulation of AmB in human skin was 40 times higher when applied as AmB-UDL than as AmBisome. AmB-UDL provided a profound AmB penetration toward deep epithelial layers, achieved without classical permeation enhancers. Because of that, topical treatments of cutaneous fungal infection and leishmaniasis with AmB-UDL may be regarded of potential of clinical significance.