Aspergillus fumigatus and Pseudomonas aeruginosa are the most prevalent fungal and bacterial pathogens associated with cystic fibrosis-related infections, respectively. P. aeruginosa eventually predominates as the primary pathogen, though it is unknown why this is the case. Label-free quantitative (LFQ) proteomics was employed to investigate the cellular response of the alveolar epithelial cell line, A549, to co-exposure of A. fumigatus and P. aeruginosa. These studies revealed a significant increase in the rate of P. aeruginosa proliferation where A. fumigatus was present. Shotgun proteomics performed on A549 cells exposed to either A. fumigatus or P. aeruginosa or to A. fumigatus and P. aeruginosa sequentially, revealed distinct changes to the host-cell proteome in response to either or both pathogens. While key signatures of infection were retained amongst all pathogen-exposed groups, including changes in mitochondrial activity and energy output, the relative abundance of proteins associated with endocytosis, phagosomes and lysosomes were decreased in sequentially exposed cells compared to cells exposed to either pathogen. Our findings indicate that A. fumigatus render A549 cells unable to internalize bacteria, thus providing an environment in which P. aeruginosa can proliferate. This research provides novel insights into the whole-cell proteomic response of A549 cells to A. fumigatus and P. aeruginosa, and highlights distinct differences in the proteome following sequential exposure to both pathogens, which may explain why P. aeruginosa can predominate.