Zika virus (ZIKV) infection has been linked to congenital defects in fetuses and infants, as exemplified by the microcephaly epidemic in Brazil. Given the overlapping presence of Dengue virus (DENV) in the majority of ZIKV epidemic regions, advanced diagnostic approaches need to be evaluated to establish the role of pre-existing DENV immunity in ZIKV infection. From 2015 to 2017, five pregnant women with suspected ZIKV infection were investigated in Pavia, Italy. Among the five pregnant women, three were DENV-ZIKV immunologically cross-reactive, and two were DENV-naïve. Advanced diagnosis included the following: (i) NS1 blockade-of-binding (BOB) ELISA assay for ZIKV specific antibodies and (ii) ELISpot assay for the quantification of effector memory T cells for DENV and ZIKV. These novel assays allowed to distinguish between related flavivirus infections. The three DENV-experienced mothers did not transmit ZIKV to the fetus, while the two DENV-naive mothers transmitted ZIKV to the fetus. Pre-existing immunity in DENV experienced mothers might play a role in cross-protection.