Cutaneous fungal and parasitic diseases remain challenging to treat, as available therapies are unable to permeate the skin barrier. Thus, treatment options rely on systemic therapy, which fail to produce high drug local concentrations but can lead to significant systemic toxicity. Amphotericin B (AmB) is highly efficacious in the treatment of both fungal and parasitic diseases such as cutaneous leishmaniasis, but is only reserved for parenteral administration in patients with severe pathophysiology. Here, we have designed and optimised AmB transfersomes [93.5 % encapsulation efficiency, size of 150 nm, and good colloidal stability (-35.02 mV)] that can remain physicochemically stable (>90 % drug content) at room temperature and 4 °C over 6 months when lyophilised and stored under desiccated conditions. AmB transfersomes possessed good permeability across mouse skin (4.91 ± 0.41 μg/cm2/h) and 10-fold higher permeability across synthetic Strat-M® membranes. In vivo studies after a single topical application in mice showed permeability and accumulation within the dermis (>25 μg AmB /g skin at 6 h post-administration) indicating the delivery of therapeutic amounts of AmB for mycoses and cutaneous leishmaniasis, while a single daily administration in Leishmania (Leishmania) amazonensis infected mice over 10 days resulted in excellent efficacy (98 % reduction in Leishmania parasites). Combining the application of AmB transfersomes with metallic microneedles in vivo increased levels in the SC and dermis but is unlikely to elicit transdermal levels. In conclusion, AmB transfersomes are promising and stable topical nanomedicines that can be readily translated for parasitic and fungal infectious diseases.