Enterovirus A71 (EV-A71) and enterovirus D68 (EV-D68) are increasingly recognized as causative agents of severe neurological complications such as acute flaccid myelitis (AFM). However, the molecular mechanisms underlying their neurovirulence and impact on neuromuscular integrity remain poorly understood. In this study, we employed human induced pluripotent stem cell-derived neuromuscular organoids (NMOs) as a model to investigate the cellular tropism and pathologic effects of EV-A71 and EV-D68. Our results demonstrate that both viruses can efficiently infect neuronal populations within NMOs, with EV-A71 exhibiting higher infectivity compared to EV-D68. Infection resulted in increased expression of cleaved caspase-3, indicative of apoptosis. We also observed stochastic cleavage of SNAP25 and SNAP29, which are two key SNARE proteins involved in synaptic vesicle fusion and autophagy, respectively. Transcriptomic analyses revealed downregulation of neuronal and muscular gene networks, with EV-A71 preferentially affecting neuronal pathways and EV-D68 having a greater impact on muscular gene expression. These findings are consistent with previous reports of virus-specific cellular tropism and suggest that distinct mechanisms of neuromuscular impairment might occur. Our study establishes NMOs as a robust platform for dissecting host–virus interactions relevant to AFM and provides novel insights into the molecular pathogenesis of EV-induced neuromuscular dysfunction, with potential implications for the development of targeted therapeutic strategies.