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Storm Daniel, the deadliest Mediterranean tropical-like cyclone, caused significant flooding in Thessaly Region, Greece, from September 4 to 7, 2023. This study examines the potential impact of such extreme weather events on vector-borne disease transmission by assessing changes in mosquito populations and West Nile virus (WNV) circulation before and after the flood in two regional units of Thessaly. Systematic monitoring data on mosquito larvae and adults, along with WNV circulation in mosquitoes and humans from 2021 to 2023, were analyzed using a weekly interrupted time series regression design controlling for confounding drivers and temporal trends. Results indicate a significant post-flood increase in Culex mosquito populations over the 7 weeks following the event. However, despite this increase—alongside optimal temperature conditions and pre-flood amplification of WNV—no corresponding rise in WNV circulation was observed in mosquitoes or human cases. This unexpected outcome may be influenced by multiple ecological factors, including disruptions of avian host communities, human displacement, and the timing of the flood during the autumn bird migration period. These findings underscore the complexity of vector-virus-host interactions and highlight the importance of continued systematic entomological surveillance for targeted mosquito control practices.