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Abstract Greener methods for the synthesis of various nanostructures with well-organized characteristics and biomedical applicability have demonstrated several advantages, including simplicity, low toxicity, cost-effectiveness, and eco-friendliness. Spinel nickel ferrite (NiFe2O4) nanowhiskers with rod-like structures were synthesized using a simple and green method; these nanostructures were evaluated by X-ray diffraction analysis, transmission electron microscopy, scanning electron microscopy, and X-ray energy diffraction spectroscopy. Additionally, the prepared nanowhiskers could significantly reduce the survival of Leishmania major promastigotes, at a concentration of 500 μg/mL; the survival of promastigotes was reduced to ≃ 26%. According to the results obtained from MTT test (in vitro), it can be proposed that further studies should be conducted to evaluate anti-leishmaniasis activity of these types of nanowhiskers in animal models.