Influenza poses a global health threat. With drug-resistant strains emerging, there is an urgent need for effective antiviral drugs. This study explores antiviral potential of flavonoids against influenza A virus (IAV) and their mechanism of action. By utilizing in silico docking as a screening approach, diosmin, orientin, and fisetin were identified as flavonoids with the strongest interactions with viral proteins. Out of them, diosmin was found to effectively inhibit IAV replication in vitro, particularly at the attachment and post-entry stages, with significant inhibition observed at 0-h post-infection (hpi) and 2 hpi, while also demonstrated prophylactic activity, peaking at - 2 hpi. Following that, diosmin significantly increases the expression of antiviral genes, which may relate to the discovery of its prophylactic activity. Proteomics analysis showed that diosmin treatment during the post-entry stage of IAV replication reduced viral protein levels, confirming its antiviral activity at this point. Additionally, diosmin also modulated host proteins related to innate immunity, inducing type I interferon and anti-inflammatory responses during the infection. These findings provide preliminary evidence of diosmin's antiviral and prophylactic activity against IAV, paving the way for further research on its mechanism of action.