"Pseudomonas aeruginosa and Aspergillus fumigatus frequently co-occur in cystic fibrosis (CF) lungs, where their interactions can alter virulence. Lipopolysaccharide (LPS), a complex glycolipid of the P. aeruginosa (PAO1) outer membrane, protects against host defences, mediates host-pathogen interactions, and acts as a potent endotoxin. This study examines how A. fumigatus secreted products impact LPS expression and surface phenotypes in P. aeruginosa. PAO1 cultures were treated with sterile-filtered 72-hour A. fumigatus supernatant (AFsn). RNA sequencing used Illumina NextSeq 2000. LPS profiles were analysed by SDS-PAGE with Pro-Q Emerald 300 staining and quantified via ImageJ. Total LPS was measured by Purpald assay, and lipid A by ELISA. Structural changes were assessed by ¹H NMR spectroscopy. Cell surface charge was measured by cytochrome c binding. Motility phenotypes were assessed on agar media. RNA-seq revealed significant upregulation of LPS biosynthesis genes (wzz, wzy, wzx, wbpB, wbpG, wbpH, hisH2, hisF2). SDS-PAGE demonstrated significant increases across O-antigen regions: short-chain (9.29%, p = 0.0157), long-chain (8.6%, p = 0.049), very long-chain (12.59%, p = 0.0172), and core regions: uncapped (16.36%, p = 0.029), capped (20.61%, p = 0.0475), core+1 (16.5%, p = 0.038). Total LPS content increased 69.9% (p < 0.0001) and lipid A by 12.4% (p = 0.0007). NMR detected subtle structural differences. Cell surface charge decreased 1.3% (p = 0.014). Motility assays revealed decreased twitching and swimming with increased swarming. A. fumigatus supernatant induces structural and compositional changes in P. aeruginosa LPS alongside altered surface properties and motility, potentially contributing to bacterial virulence in CF lungs."