Pseudomonas aeruginosais a model quorum sensing (QS) pathogen with three interconnected QS circuits that control the production of virulence factors and antibiotic tolerant biofilms. ThepqsQS system ofP. aeruginosais responsible for the biosynthesis of diverse 2-alkyl-4-quinolones (AQs), of which 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) function as QS signal molecules. Transcriptomic analyses revealed that HHQ and PQS influenced the expression of multiple genes via PqsR-dependent and -independent pathways whereas 2-heptyl-4-hydroxyquinolineN-oxide (HQNO) had no effect onP. aeruginosatranscriptome. HQNO is a cytochromebc1inhibitor that causesP. aeruginosaprogrammed cell death and autolysis. However,P. aeruginosa pqsLmutants unable to synthesize HQNO undergo autolysis when grown as colony biofilms. The mechanism by which such autolysis occurs is not understood. Through the generation and phenotypic characterization of multipleP. aeruginosaPAO1 mutants producing altered levels of AQs in different combinations, we demonstrate that mutation ofpqsLresults in the accumulation of HHQ which in turn leads to Pf4 prophage activation and consequently autolysis. Notably, the effect of HHQ on Pf4 activation is not mediatedviaits cognate receptor PqsR. These data indicate that the synthesis of HQNO in PAO1 limits HHQ-induced autolysis mediated by Pf4 in colony biofilms. A similar phenomenon is shown to occur inP. aeruginosacystic fibrosis (CF) isolates, in which the autolytic phenotype can be abrogated by ectopic expression ofpqsL.