Pseudomonas aeruginosa is implicated in nosocomial infections and chronic respiratory infections in cystic fibrosis patients. Chlorhexidine diacetate (CHX) is a biguanide disinfectant used for bacterial control in the hospital and agricultural and domestic environments. A better understanding of the mechanism of action of CHX and the resulting response elicited by P. aeruginosa to CHX will facilitate its effective utilization for P. aeruginosa control in these environments. This study presents, for the first time, the transcriptomic response of P. aeruginosa to 0.008 mM CHX after 10 and 60 min. Our results reveal that, after both treatment times, membrane transport, oxidative phosphorylation, and electron transport genes were downregulated. After 10 min, DNA repair was downregulated and the oprH gene that blocks the self-promoted uptake of antimicrobials was upregulated. After 60 min, outer membrane protein, flagellum, pilus, oxidative phosphorylation, and electron transport genes were downregulated. The mexC and mexD genes of the MexCD-OprJ multidrug efflux pump were significantly upregulated after both treatment times. The results of this study improve our understanding of the mode of action of CHX on P. aeruginosa and provide insights into the mechanism of action of other biguanide disinfectants which can be used for the development of more efficient disinfectants.