Aspergillus fumigatus (Af) is the most prevalent and life-threatening airborne opportunistic fungal pathogen, particularly affecting individuals with cystic fibrosis (CF) and other chronic lung diseases. Although Af pathogenesis has been extensively studied in mono-species infections, the impact of coinfecting microbes on Af physiology during infection has remained poorly understood. Using an advanced in vitro coculture model that mimics the CF lung environment, this work integrated comparative multi-omics analyses (proteomics, transcriptomics, metabolomics) and time-lapse confocal microscopy with reverse genetics to investigate Af physiology in the presence and absence of the bacterial pathogen Pseudomonas aeruginosa (Pa). These studies identified two major mechanisms of interkingdom communication: the fungal secondary metabolite gliotoxin, produced by Af, and the bacterial metabolite hydrogen cyanide, produced by Pa. Mechanistic analyses revealed distinct physiological and metabolic shifts in both organisms during coculture in synthetic CF sputum medium. In addition, parallel experiments using clinical isolates of Af and Pa demonstrated that these secondary metabolite–mediated response networks are maintained following chronic infection. Using deletion mutants in biosynthetic and defensive genes for the above toxins, we also uncovered the functions of previously uncharacterized genes central to interkingdom microbial interactions in both organisms. Collectively, these findings establish a mechanistic framework for studying fungal–bacterial cross-talk in vitro and demonstrate how secondary metabolite exchange shapes microbial physiology within polymicrobial respiratory environments.