Staphylococcus aureus (S. aureus) is a bacterial pathogen that accounts for a significant number of serious nosocomial and community-acquired infections. The notoriety of this pathogen stems from its ability to produce a plethora of virulence factors, including a collection of bi-component, pore-forming leukotoxins. Strains associated with human infections can produce up to five leukotoxins known as LukSF-PV, LukAB, HlgAB, HlgCB, and LukED, which target and kill immune cells required to mount effective innate and adaptive immune responses. Among the leukotoxins, LukSF-PV, LukED, and HlgACB share more than 65% amino acid sequence identity, which in turn facilitates the formation of “non-natural” toxin complexes (e.g. LukS-PV/LukD, LukE/LukF-PV) that can also target and kill leukocytes. By employing a structure-function based approach, we have identified mutations in LukE and LukD that abolish LukED’s ability to kill primary human neutrophils. Interestingly, when mixed with wild type LukED, the mutant toxin subunits block the cytolytic activity of LukED in a dominant-negative manner. Introduction of similar mutations into LukSF-PV, HlgAB, and HlgCB also render these toxins dominant-negative against their wild type counterparts. Remarkably, the dominant-negative mutant toxins also exhibit cross-inhibitory activity in vitro. Lastly, we show that the dominant-negative toxins attenuate S. aureus using ex vivo and in vivo models of infection. Taken together, this study highlights the therapeutic potential of targeting S. aureus leukotoxins as a strategy to attenuate this important human pathogen.