Development of anti-influenza A virus (IAV) drugs with novel targets and low toxicity is critical for preparedness against influenza outbreaks. In the current study, our results indicated that the novel polyketide compound purpurquinone B (PPQ-B) derived from acid-tolerant fungus Penicillium purpurogenum strain JS03-21 suppressed the replication of IAV in vitro with low toxicity, and may block some stages after virus adsorption. PPQ-B could inhibit H1N1 (A/Puerto Rico/8/34; PR8), H1N1 (A/California/04/2009; Cal09) and H3N2 (A/swine/Minnesota/02719/2009) virus replication in vitro, suggesting that PPQ-B possesses broad-spectrum anti-IAV activities. PPQ-B's antiviral activity may be largely related to its inhibition of some steps that occur 0–4 h after adsorption. Oral administration of PPQ-B could decrease pulmonary viral titers and improve survival rate in IAV infected mice. PPQ-B also significantly decreased the production of inflammatory factors TNF-α, IL-6, RANTES and KC in IAV infected lungs and A549 cells, suggesting that PPQ-B may also attenuate the inflammatory responses caused by IAV infection. PPQ-B may down-regulate the NF-κB and MAPK pathways to inhibit both virus replication and inflammatory responses. In summary, PPQ-B has the potential to be developed into a novel anti-IAV drug targeting host EGFR pathway in the future.