Leishmania braziliensis and Leishmania amazonensis are both causative agents of cutaneous leishmaniasis in South America. However, patient prognosis and the host immune response differ considerably depending on the infecting parasite species. The mechanisms underlying these differences appear to be multifactorial, with both host and parasite components contributing to disease outcome. As neutrophils are a prominent component of the inflammatory infiltrate in chronic cutaneous, diffuse cutaneous and mucocutaneous lesions, we examined neutrophil activation and microbicidal activity against amastigotes of L. amazonensis and L. braziliensis. We found that murine neutrophils internalized L. braziliensis amastigotes with greater efficiency than did L. amazonensis amastigotes. Additionally, L. braziliensis infection was a potent trigger for neutrophil activation, oxidative burst, degranulation and the production of interleukin (IL)-22 and IL-10, while L. amazonensis amastigotes poorly induced these responses. Finally, neutrophils were able to kill L. braziliensis amastigotes, especially when cells were activated with phorbol myristate acetate. L. amazonensis amastigotes, however, were highly resistant to neutrophil microbicidal mechanisms. This study reveals, for the first time, differential neutrophil responsiveness to distinct species of Leishmania amastigotes and highlights the complexity of neutrophil-amastigote interactions during chronic leishmaniasis.