Streptococcus pneumoniae rapidly kills Staphylococcus aureus by producing membrane-permeable hydrogen peroxide (H2O2). The mechanism by which S. pneumoniae-produced H2O2 mediates S. aureus killing was investigated. An in vitro model that mimicked S. pneumoniae-S. aureus contact during colonization of the nasopharynx demonstrated that S. aureus killing required outcompeting densities of S. pneumoniae. Compared to the wild-type strain, isogenic S. pneumoniae ΔlctO and S. pneumoniae ΔspxB, both deficient in production of H2O2, required increased density to kill S. aureus. While residual H2O2 activity produced by single mutants was sufficient to eradicate S. aureus, an S. pneumoniae ΔspxB ΔlctO double mutant was unable to kill S. aureus. A collection of 20 diverse methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains showed linear sensitivity (R2 = 0.95) for S. pneumoniae killing, but the same strains had different susceptibilities when challenged with pure H2O2 (5 mM). There was no association between the S. aureus clonal complex and sensitivity to either S. pneumoniae or H2O2. To kill S. aureus, S. pneumoniae produced ∼180 μM H2O2 within 4 h of incubation, while the killing-defective S. pneumoniae ΔspxB and S. pneumoniae ΔspxB ΔlctO mutants produced undetectable levels. Remarkably, a sublethal dose (1 mM) of pure H2O2 incubated with S. pneumoniae ΔspxB eradicated diverse S. aureus strains, suggesting that S. pneumoniae bacteria may facilitate conversion of H2O2 to a hydroxyl radical ( OH). Accordingly, S. aureus killing was completely blocked by incubation with scavengers of OH radicals, dimethyl sulfoxide (Me2SO), thiourea, or sodium salicylate. The OH was detected in S. pneumoniae cells by spin trapping and electron paramagnetic resonance. Therefore, S. pneumoniae produces H2O2, which is rapidly converted to a more potent oxidant, hydroxyl radicals, to rapidly intoxicate S. aureus strains.