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The physicochemical and microbiological stability of a hyaluronic acid-based nanostructured topical delivery system containing P. pubescens fruit oil was evaluated, and the in vitro antileishmanial activity of the nanoemulsion against Leishmania amazonensis and the cytotoxicity on macrophages was investigated. The formulation stored at 5 ± 2 °C, compared with the formulation stored at 30 and 40 ± 2 °C, showed a higher chemical and physical stability during the period analyzed and in the accelerated physical stability study. The formulation stored at 40 °C presented a significant change in droplet diameter, polydispersity index, zeta potential, pH, active compound, and consistency index and was considered unstable. The microbiological stability of the formulations was confirmed. The leishmanicidal activity of the selected system against intracellular amastigotes was significantly superior to that observed for the free oil. However, further research is needed to explore the use of the hyaluronic acid-based nanostructured system containing P. pubescens fruit oil for the treatment of cutaneous leishmaniasis.