Tick-Borne Encephalitis virus (TBEV) and West Nile virus (WNV) are two Flaviviruses 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 order to have more physiologically relevant modeIs, we recently used human neuronal/glial cells in culture and demonstrated their permissivity to TBEV and WNV (see poster François Piumi). Here, with the aim of continuing to develop ever more relevant in vitro models, we produced and characterized three-dimensional (3D) cellular models, called Brain Organoids (BOs), which mimic the tissue organization of the human and equine central nervous system. We infected human BOs (hBOs) with TBEV and WNV and equine BOs (eBOs) with WNV and assessed viral replication through quantification of viral RNA (RT-qPCR) and viral particles (TCID 50) in supernatants, and immunostaining with antibodies specific to the viruses on 10 µm sections. Both viruses replicated efficiently in hBOs, as did WNV in eBOs. Infected hBOs and eBOs were next used to test the antiviral activity of chemical compounds, some of which having a dose-dependent activity. Thus, human and equine BOs represent novel, physiologically relevant, 3D in vitro models of viral infection which can be used to confirm antiviral activity of chemical compounds before in vivo testing.