Background: Targeting cells of the host immune system is a promising approach to fight against Influenza A virus (IAV) infection. Macrophage cells use the NADPH oxidase-2 (NOX2) enzymatic complex as a first line of defense against pathogens by generating superoxide ions O-2(-) and releasing H2O2. Herein, we investigated whether targeting membrane-embedded NOX2 decreased IAV entry via raft domains and reduced inflammation in infected macrophages. Methods: Confocal microscopy and western blots monitored levels of the viral nucleoprotein NP and p67(Phox), NOX2 activator subunit, Elisa assays quantified TNF-alpha levels in LPS or IAV-activated mouse or porcine alveolar macrophages pretreated with a fluorescent NOX inhibitor, called nanoshutter NS1. Results: IAV infection in macrophages promoted p67(Phox) translocation to the membrane, rafts clustering and activation of the NOX2 complex at early times. Disrupting rafts reduced intracellular viral NP. NS1 markedly reduced raft clustering and viral entry by binding to the C-terminal of NOX2 also characterized in vitro. NS1 decrease of TNF-a release depended on the cell type. Conclusion: NOX2 participated in IAV entry and raft-mediated endocytosis. NOX2 inhibition by NS1 reduced viral entry. NS1 competition with p67(Phox) for NOX2 binding shown by in silico models and cell-free assays was in agreement with NS1 inhibiting p67(Phox) translocation to membrane-embedded NOX2 in mouse and porcine macrophages. General significance: We introduce NS1 as a compound targeting NOX2, a critical enzyme controlling viral levels and inflammation in macrophages and discuss the therapeutic relevance of targeting the C-terminal of NADPH oxidases by probes like NS1 in viral infections.