Pseudomonas aeruginosa and Staphylococcus aureus cause debilitating polymicrobial infections in diverse patient populations. Studies of these bacterial pathogens in coculture have shown that environmental variables including Fe availability and the host-defense protein calprotectin (CP) impact coculture dynamics. To decipher how CP modulates interactions between P. aeruginosa and S. aureus, we employed dual-species RNA-seq to examine the transcriptional responses of both pathogens in coculture to CP treatment and metal depletion. Analysis of these responses revealed that, for both P. aeruginosa and S. aureus, CP treatment not only induced gene expression changes consistent with single- and multi-metal starvation responses, but also induced gene expression changes that were not observed under metal limitation. For P. aeruginosa, CP treatment induced gene expression changes pointing to a shift in chorismate flux away from alkylquinolone and phenazine biosynthesis towards folate biosynthesis. These observations were consistent with decreased production of alkylquinolones by P. aeruginosa, including the potent anti-staphylococcal alkylquinolone N-oxides. CP treatment afforded perturbed levels of two quorum sensing molecules, 3-oxo-C12-homoserine lactone and C4-homoserine lactone, produced by P. aeruginosa. In addition, CP treatment enhanced the ability of S. aureus to mount Fe starvation responses and caused S. aureus to express host virulence genes. This analysis illuminated physiological consequences of CP treatment that extend beyond metal starvation and that these consequences impact interspecies interactions. Our findings provide a working model in which CP effectively disarms P. aeruginosa by inhibiting the production of anti-staphylococcal factors and boosts the ability of S. aureus to protect itself from attack.