A new spirocyclic γ-lactam, named spirostaphylotrichin X (1), and three related known spirostaphylotrichins (2-4) were isolated from the marine-derived fungus Cochliobolus lunatus SCSIO41401. Their structures were determined by spectroscopic analyses. Spirostaphylotrichin X (1) displayed obvious inhibitory activities against multiple influenza virus strains, with IC50 values from 1.2 to 5.5 μM. Investigation of the mechanism showed that 1 inhibited viral polymerase activity and interfered with the production of progeny viral RNA. Homogeneous time-resolved fluorescence, surface plasmon resonance assays, and a molecular docking study revealed that 1 could inhibit polymerase PB2 protein activity by binding to the highly conserved region of the cap-binding domain of PB2. These results suggest that 1 inhibits the replication of influenza A virus by interfering with the activity of PB2 protein and that 1 represents a new type of potential lead compound for the development of anti-influenza therapeutics.