Tick-Borne Encephalitis virus (TBEV) and West Nile virus (WNV) are two Flavivirus that can cause severe and potentially fatal neurological disorders in human and/or horses, for which there is currently no antiviral treatment. These viruses have mostly been studied in cell lines, often not representative of their natural tropism. In this study, we compared the antiviral activity of 9 molecules against TBEV in 3 cellular models of different level of physiological relevance: a human pulmonary cell line (A549), human neural progenitor cells (hNPCs) and human neuronal/glial cells (hNGCs). We showed that most of the molecules had an antiviral activity in A549 but only three were efficient in hNGCs, demonstrating the importance of physiologically relevant models. Recently, three-dimensional (3D) cellular models called brain organoids (BOs) have been developed with the aim of better representing the tissue organisation of the CNS. We infected human BOs with TBEV and WNV and equine BOs with WNV and assessed viral replication through quantification of viral RNA (RT-qPCR) and viral particles (TCID50) in supernatants. Both viruses replicated efficiently in hBOs, as did WNV in eBOs. They represent novel, physiologically relevant, 3D in vitro models of infection. eBOs were then used to test the antiviral activity of 3 molecules. One molecule caused a dose-dependent decrease of viral particles in eBOs supernatants, showing the potential of these models to confirm antiviral activity of compounds before in vivo testing.