© 2020 Margalit et al. Individuals with cystic fibrosis are susceptible to co-infection by Aspergillus fumigatus and Pseudomonas aeruginosa Despite the persistence of A. fumigatus in the cystic fibrosis lung P. aeruginosa eventually predominates as the primary pathogen. Several factors are likely to facilitate P. aeruginosa colonization in the airways, including alterations to the microbial environment. The cystic fibrosis airways are hypoxic, nitrate-rich environments, and the sputum has higher amino acid concentrations than normal. In this study, significant growth proliferation was observed in P. aeruginosa when the bacteria were exposed to A. fumigatus culture filtrates (CuF) containing a high nitrate content. Proteomic analysis of the A. fumigatus CuF identified a significant number of environment-altering proteases and peptidases. The molecular mechanisms promoting bacterial growth were investigated using label-free quantitative (LFQ) proteomics to compare the proteome of P. aeruginosa grown in the A. fumigatus CuF and in CuF produced by a P. aeruginosa-A. fumigatus co-culture, to that cultured in P. aeruginosa CuF. LFQ proteomics revealed distinct changes in the proteome of P. aeruginosa when cultured in the different CuFs, including increases in the levels of proteins involved in denitrification, stress response, replication, amino acid metabolism and efflux pumps, and a down-regulation of pathways involving ABC transporters. These findings offer novel insights into the complex dynamics that exist between P. aeruginosa and A. fumigatus Understanding the molecular strategies that enable P. aeruginosa to predominate in an environment where A. fumigatus exists is important in the context of therapeutic development to target this pathogen.