© 2015, American Society for Microbiology. The oral cavity harbors a diverse community of microbes that are physiologically unique. Oral microbes that exist in this polymicrobial environment can be pathogenic or beneficial to the host. Numerous oral microbes contribute to the formation of dental caries and periodontitis; however, there is little understanding of the role these microbes play in systemic infections. There is mounting evidence that suggests that oral commensal streptococci are cocolonized with Pseudomonas aeruginosa during cystic fibrosis pulmonary infections and that the presence of these oral streptococci contributes to improved lung function. The goal of this study was to examine the underlying mechanism by which Streptococcus parasanguinis antagonizes pathogenic P. aeruginosa. In this study, we discovered that oral commensal streptococci, including Streptococcus parasanguinis, Streptococcus sanguinis, and Streptococcus gordonii, inhibit the growth of P. aeruginosa and that this inhibition is mediated by the presence of nitrite and the production of hydrogen peroxide (H2O2) by oral streptococci. The requirement of both H2O2 and nitrite for the inhibition of P. aeruginosa is due to the generation of reactive nitrogenous intermediates (RNI), including peroxynitrite. Transposon mutagenesis showed that a P. aeruginosa mutant defective in a putative ABC transporter permease is resistant to both streptococcus/nitrite- and peroxynitrite-mediated killing. Furthermore, S. parasanguinis protects Drosophila melanogaster from killing by P. aeruginosa in a nitrite-dependent manner. Our findings suggest that the combination of nitrite and H2O2 may represent a unique anti-infection strategy by oral streptococci during polymicrobial infections.