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ABSTRACT Every year, dengue virus (DENV) causes one hundred million infections worldwide that can result in dengue disease and severe dengue. Two other mosquito-borne flaviviruses, i.e., Zika virus (ZIKV) and West Nile virus (WNV), are responsible of prolonged outbreaks and are associated with severe neurological diseases, congenital defects, and eventually death. These three viruses, despite their importance for global public health, still lack specific drug treatments. Here, we describe the structure-guided discovery of small molecules with pan-flavivirus antiviral potential by a virtual screening of ~1 million structures targeting the NS3-NS5 interaction surface of different flaviviruses. Two molecules inhibited the interaction between DENV NS3 and NS5 in vitro and the replication of all DENV serotypes as well as ZIKV and WNV and exhibited low propensity to select resistant viruses. Remarkably, one molecule demonstrated efficacy in a mouse model of dengue by reducing peak viremia, viral load in target organs, and associated tissue pathology. This study provides the proof of concept that targeting the flaviviral NS3-NS5 interaction is an effective therapeutic strategy able to reduce virus replication in vivo and discloses new chemical scaffolds that could be further developed, thus providing a significant milestone in the development of much awaited broad-spectrum antiflaviviral drugs. IMPORTANCE More than one-third of the human population is at risk of infection by different mosquito-borne flaviviruses. Despite this, no specific antiviral drug is currently available. In this work, using a computational approach based on molecular dynamics simulation and virtual screening of ~1 million small-molecule structures, we identified a compound that targets the interaction between the two sole flaviviral enzymes, i.e., NS3 and NS5. This compound demonstrated pan-serotype anti-DENV activity and pan-flavivirus potential in infected cells, low propensity to select viral resistant mutant viruses, and efficacy in a mouse model of dengue. Broad-spectrum antivirals are much awaited, and this work represents a significant advance toward the development of therapeutic molecules with extended antiflavivirus potential that act by an innovative mechanism and could be used alone or in combination with other antivirals.