Plant respiratory burst oxidase homologs are prominent sources of reactive oxygen species (ROS) in signal transduction and in interaction with microbes. However, the function of respiratory burst oxidase homologue (RBOH) genes in interaction with microbes might differ for certain plant and pathogen species. We produced transgenic barley knock down (KD) for the HvRBOHF2 isoform of NADPH oxidases. Young HvRBOHF2 KD shoots did not show obvious morphological alterations from the wild type but adult HvRBOHF2 KD plants developed fewer tillers, were less fertile, and showed spontaneous cell death in leaf mesophyll. Additionally, HvRBOHF2 KD plants were unable to contain wound-induced cell death. Before developmental failure became obvious, young HvRBOHF2 KD seedlings were much more susceptible to penetration by the biotrophic powdery mildew fungus Blumeria graminis f. sp. hordei. Strikingly, the B. graminis f. sp. hordei-induced cell-wall-associated oxidative burst was not substantially attenuated in HvRBOHF2 KD plants but enhanced susceptibility apparently influenced the subcellular site of hydrogen peroxide accumulation. Taken together, misexpression of HvRBOHF2 caused failure of barley to normally develop penetration resistance to B. graminis f. sp. hordei and to control leaf cell death.