Significance Mucosal barrier tissues participate in immune defense in infections, but NADPH oxidases expressed in these epithelia are much less efficient in their oxidant output than the phagocyte oxidase. The importance of releasing low hydrogen peroxide (H 2 O 2 ) concentrations as a host defense mechanism against pathogens remains unclear. Here, we demonstrate that nano to submicromolar H 2 O 2 disrupts the tyrosine phosphorylation network in several pathogens by an oxidative dephosphorylation process; this is accomplished by irreversible chemical modification of key phosphotyrosine residues, which in turn changes protein activity without affecting bacterial viability. This process is a host-initiated antivirulence strategy, reducing the fitness of pathogens in the extracellular space.