Fusarium graminearum, which causes Fusarium head blight (FHB) in cereals, is one of the most devastating fungal diseases by causing great yield losses and mycotoxin contamination. A major bioactive ingredient, venturicidin A (VentA), was isolated from Streptomyces pratensis S10 mycelial extract with an activity-guided approach. No report is available on antifungal activity of VentA against F. graminearum and effects on deoxynivalenol (DON) biosynthesis. Here, VentA showed a high antagonistic activity toward F. graminearum with an EC50 value of 3.69 μg/mL. As observed by scanning electron microscopy, after exposure to VentA, F. graminearum conidia and mycelia appeared abnormal. Different dyes staining revealed that VentA increased cell membrane permeability. In growth chamber and field trials, VentA effectively reduced disease severity of FHB. Moreover, VentA inhibited DON biosynthesis by reducing pyruvic acid, acetyl-CoA production, and accumulation of reactive oxygen species (ROS) and then inhibiting trichothecene (TRI) genes expression and toxisome formation. These results suggest that VentA is a potential fungicide for controlling FHB.