There is an urgent need for safe, efficacious, affordable and field-adapted drugs for the treatment of cutaneous leishmaniasis, a disease which affects around 1.5 million people worldwide every year. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, anti-leishmanial and immunostimulatory activities. The work described here found that chitosan and its derivatives were approximately 7-20 times more active in vitro against Leishmania promastigotes and amastigotes at pH 6.5 than at pH 7.5, with high molecular weight chitosan being the most potent. Despite the in vitro activation of bone marrow macrophages by chitosan to produce nitric oxide and reactive oxygen species, this work showed that the anti-leishmanial activity of chitosan was not mediated by these metabolites. It was subsequently shown that rhodamine-labelled chitosan is taken up by pinocytosis and accumulates in the parasitophorous vacuole of Leishmania-infected macrophages. The application of chitosan in drug delivery systems was then studied by preparing two types of chitosan nanoparticles (positive (with tripolyphosphate sodium (TPP)) and negative (with dextran sulphate) surface charge with different sizes) and incorporation of amphotericin B within these nanoparticles. These amphotericin B-loaded nanoparticles demonstrated a good in vitro anti-leishmanial activity, similar to pure amphotericin B, and were also significantly less toxic than pure amphotericin B. The positive amphotericin B-loaded chitosan-TPP nanoparticles showed promising in vivo efficacy against cutaneous leishmaniasis caused by L. major in the BALB/c mouse model, via the intravenous route, and they were more active than AmBisome®. The impact of an in vitro media perfusion system on host cell phagocytosis and macropinocytosis was evaluated as well as the anti-leishmanial activity of chitosan solution and blank or amphotericin B-loaded chitosan-TPP nanoparticles. There was a significant difference between in vitro static and flow culture systems in the cell uptake and anti-leishmanial activity of the studied compounds.