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Extracellular vesicles (EVs) released from pathogenic protozoans play crucial roles in host–parasite communication and disease pathogenesis. <i>Naegleria fowleri</i> is a free-living protozoan causing primary amoebic meningoencephalitis, a fatal disease in the central nervous system. This study aims to explore the roles of <i>N. fowleri</i>-derived EVs (<i>Nf</i>-EVs) in host–pathogen interactions using the THP-1 cell line as a model. The <i>Nf</i>-EVs were isolated from the <i>N. fowleri</i> trophozoite culture supernatant using sequential centrifugation and characterized by nanoparticle tracking analysis and transmission electron microscopy. The functional roles of <i>Nf</i>-EVs in the apoptosis and immune response induction of THP-1 monocytes and macrophages were examined by flow cytometry, quantitative PCR, and ELISA. Results showed that <i>Nf</i>-EVs displayed vesicles with bilayer membrane structure approximately 130–170 nm in diameter. The <i>Nf</i>-EVs can be internalized by macrophages and induce macrophage responses by induction of the expression of costimulatory molecules CD80, CD86, HLA-DR, and CD169 and the production of cytokine IL-8. However, <i>Nf</i>-EVs did not affect the apoptosis of macrophages. These findings illustrate the potential role of <i>Nf</i>-EVs in mediating the host immune cell activation and disease pathogenesis.