Amphotericin B (AmB) is a polyene macrolide antifungal agent that binds to ergosterol, disrupting fungal membranes through lipid interactions. However, its effects on host cell protein composition remain unclear. In this study, A549 cells were pretreated with AmB prior to infection with the influenza A virus, and early and late endosomal fractions were isolated. DIA-based quantitative proteomics revealed that AmB alters the protein composition of endosomes during infection, suggesting that it modulates endosomal protein dynamics in virus-infected cells.