Recent advances in the understanding of neuropathogenesis associated with Zika virus (ZIKV) infection has led to descriptions of neonatal microcephaly cases. However, none of these reports have evaluated the humoral response during ZIKV infection. We report here polyfunctional immune activation associated with increased interferon‐gamma‐inducible protein 10, interleukin (IL)‐6, IL‐8, vascular endothelial growth factor (VEGF), monocyte chemoattractive protein 1 (MCP‐1), and granulocyte colony‐stimulating factor (G‐CSF) levels in the amniotic fluid of ZIKV‐positive pregnant women with neonatal microcephaly. These cytokines have been associated not only with neuronal damage, but also with differentiation and proliferation of neural progenitor cells. Our results suggested that the immune activation caused by ZIKV infection in the uterine environment could also interfere with fetal development. ANN NEUROL 2017;81:152–156