Fusarium head blight (FHB) is among the chief threats to profitable barley production, and fungicide applications are one of two main strategies for reducing FHB damage to barley crops. However, there is very little published information on optimal timing of such applications. A 4-year field study was conducted with winter barley in Raleigh, North Carolina, to compare three timings for fungicide application: 50% spike emergence (Zadoks growth stage or GS 55), 100% spike emergence (GS 59), and 6 days after GS 59. Three winter barley cultivars with varying levels of FHB resistance were grown for four successive years (2018 to 2021) in a split-plot experiment and inoculated each spring with Fusarium-infected corn spawn. Three fungicides were compared: propiconazole + pydiflumetofen (Miravis Ace), prothioconazole + tebuconazole (Prosaro), and metconazole (Caramba). Correlations among visual symptoms and assays of harvested grain were modest and were weakened by fungicide applications. Across years and cultivars, deoxynivalenol (DON) and percent Fusarium-infected kernels were most reduced relative to the nontreated control by fungicide applications at the latest timing (GS 59 + 6 days). The early (GS 55) timing resulted in DON not significantly different from the nontreated control. Based on these results, it is recommended that to minimize damage from FHB, fungicide should be applied to winter barley several days after GS 59 (100% spike emergence), and not before GS 59.