Staphylococcus aureus (S. aureus) is a human pathogen that relies on the subversion of host phagocytes to support its pathogenic lifestyle. S. aureus strains can produce up to five beta‐barrel, bi‐component, pore‐forming leukocidins that target and kill host phagocytes. Thus, preventing immune cell killing by these toxins is likely to boost host immunity. Here, we describe the identification of glycine‐rich motifs within the membrane‐penetrating stem domains of the leukocidin subunits that are critical for killing primary human neutrophils. Remarkably, leukocidins lacking these glycine‐rich motifs exhibit dominant‐negative inhibitory effects toward their wild‐type toxin counterparts as well as other leukocidins. Biochemical and cellular assays revealed that these dominant‐negative toxins work by forming mixed complexes that are impaired in pore formation. The dominant‐negative leukocidins inhibited S. aureus cytotoxicity toward primary human neutrophils, protected mice from lethal challenge by wild‐type leukocidin, and reduced bacterial burden in a murine model of bloodstream infection. Thus, we describe the first example of staphylococcal bi‐component dominant‐negative toxins and their potential as novel therapeutics to combat S. aureus infection.