Many endemic, emerging, re-emerging or potentially emerging RNA viruses (Flavi-, Alpha-, Henipa-, Rhabdo-, Corona-viruses…) target the human central nervous system (CNS), causing severe neurological disorders, sometimes fatal or leading to debilitating consequences. The Flavivirus genus includes many of these highly neuropathogenic viruses (West Nile virus, Tick-borne encephalitis virus-TBEV, Zika virus…). Despite their dangerousness, only few vaccines exist and there is currently no available antiviral treatment. Whereas many efforts are made for the identification of antiviral molecules, including with broad-spectrum properties, most studies performed so far used models that are not physiologically relevant. This could explain the lack of antiviral activity or excessive toxicity often observed in clinical trials and their consecutive failure. Here, by comparing the antiviral activity of 8 selected molecules on 3 different models of TBEV infection of different relevance (A549, human neural progenitor cells and human neuronal/glial cells), we indeed demonstrated major differences in their capacity to inhibit viral infection: whereas most of the molecules had an antiviral activity in A549 cell line, only one of them was efficient in TBEV-infected human neuronal/glial cells (hNGCs). These results demonstrated the importance of developing physiologically relevant models for testing or screening drugs for their antiviral activity. We thus aimed at developing an image-based phenotypic screen using hNGCs differentiated from fetal human neural progenitor cells. TBEV infection of hNGCs was previously shown to reproduce major hallmarks of TBEV infection in the human brain, such as neuronal death and astrogliosis. Using this unique screen, we identified new antiviral compounds. Two of them may be therapeutically repositioned in the future as they are drugs currently used in human therapy. We next demonstrated that one of them also exerts a strong antiviral activity in a newly developed model of TBEV-infected cerebral organoids, confirming its potential interest for the treatment of human tick-borne encephalitis. We hope that, by using these physiologically relevant 2D and 3D in vitro models of TBEV infection, we will select antiviral compounds with a very high probability to be efficient and non-toxic in the human brain.