A spheroid is a cell aggregate in a three-dimensional context; thereby, it recapitulates the cellular architecture in human tissue. However, the utility of spheroids as an assay for host–parasite interactions remains unexplored. This study demonstrates the potential use of neurospheroids for assessing the cytotoxicity of the life-threatening pathogenic amoeba Balamuthia mandrillaris. The neuroblastoma SH-SY5Y cells formed a spheroid in a hanging drop of culture medium. Cellular damage caused by B. mandrillaris trophozoites on human neuronal spheroids was observed using microscopic imaging and ATP detection. B. mandrillaris trophozoites rapidly caused a decrease in ATP production in the spheroid, leading to loss of neurospheroid integrity. Moreover, 3D confocal microscopy imaging revealed interactions between the trophozoites and SH-SY5Y neuronal cells in the outer layer of the neurospheroid. In conclusion, the neurospheroid allows the assessment of host cell damage in a simple and quantitative manner.