Pseudomonas aeruginosa is the predominant bacterial species infecting the sputum and lungs of adults with cystic fibrosis (CF) and is difficult to eradicate with traditional antibiotic treatment. One of the hallmarks of P. aeruginosa colonisation is its ability to form strong biofilm matrix that inhibit antibiotic penetration. P. aeruginosa biofilm matrix primarily consists of exopolysaccharides, extracellular DNA (eDNA), and the virulence factor pyocyanin. P. aeruginosa infected CF lung secretions (sputum/mucus) contain a significant amount of pyocyanin (up to 27 µg/ml) and eDNA (3-14 mg/ml), compared to none in uninfected lung secretions. The recent finding that the P. aeruginosa metabolite pyocyanin intercalates with eDNA to strengthen the biofilm has led to a search for inhibitors of pyocyanin binding and eDNA accumulation. The antioxidant glutathione (GSH) directly binds to pyocyanin, thereby disrupting its’ association with eDNA, whilst DNase I non-specifically degrades DNA. We investigated the transcriptomic effects of GSH and DNase I on acute and chronic isogens of the Australian Epidemic strain-1 in artificial sputum medium (ASMDM) to imitate CF lung conditions. Total RNA was extracted from 48hr biofilms at 8hr post-inoculation with 2 mM GSH and 50 units DNase I, and depleted for rRNA. mRNA transcripts (n=3) were tiled and sequenced using RNA-sequencing and compared to those of a non-GSH control of the strain grown simultaneously under the same conditions. Results showed disruption of the biofilm with GSH resulted in upregulation of genes associated with acute infectivity, including pyocyanin biosynthesis, indicating that P. aeruginosa is able to adapt to removal of its biofilm by reversion from sessile to planktonic lifestyle. This is an important finding in the evaluation of combined inhaled glutathione with DNase I for treatment of chronic P. aeruginosa infections in individuals with CF.