Bacterial and fungal species within polymicrobial biofilms engage in intricate interkingdom interactions that regulate community behavior and environmental adaptation, with profound implications for human health.    We hypothesize that fungal contributions to these interactions are mediated by conserved, uncharacterized genes and signaling pathways. To address this, we use the filamentous fungus Aspergillus fumigatus (Af) as a model to study fungal-bacterial biofilm dynamics and antagonism. By dissecting the molecular signaling pathways involved, we aim to reveal how fungi sense and respond to their environment, regulate gene expression, and shape community composition within polymicrobial ecosystems.   We cultured the respiratory pathogen Af in synthetic sputum media, both alone and in co-culture with three bacterial pathogens commonly co-isolated during infection: Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cenocepacia. Temporal biofilm dynamics were monitored using confocal microscopy, and transcriptome profiling captured the fungal response to each bacterial partner. Targeted gene deletions were then used to assess the functional roles of responsive fungal genes in biofilm dynamics.   Our analysis revealed that Af displays both a ‘core’ transcriptional response to bacteria, and species-specific responses tailored to individual polymicrobial partners. We targeted responsive Af genes with no known function and observed growth deficits for mutant strains upon bacterial co-culture. Remarkably, these Af mutants also displayed clear fitness alterations when exposed to antifungals or stressors. This suggests that genes responsive to polymicrobial interactions also contribute to host-induced stress adaptation and antifungal tolerance.    In conclusion, our work identifies novel fungal genes influencing interkingdom interactions and highlights potential targets for disrupting polymicrobial infections.