Cold atmospheric-pressure plasma (CAP) is a relatively new method used for bacterial inactivation. CAP is ionized gas that can be generated by applying an electric current to air or a feeding gas. It contains reactive species and emits UV radiation, which have antibacterial activity. Previous data suggests that CAP is effective in microbial inactivation and can decontaminate and sterilize surfaces, but its exact mode of action is still under debate. This study demonstrates the effect of CAP on the whole proteome of Pseudomonas aeruginosa PAO1 biofilms, which is a dominant pathogen in cystic fibrosis and medical device-related infections. Liquid chromatography-mass spectrometry (LC-MS) was used to identify differentially regulated proteins of whole cell P. aeruginosa extracts. A total of 16 proteins were identified to be affected by plasma treatment compared to the control. Eight of the identified proteins have functions in transcription and translation and their expression changes are likely to be part of a general physiological response instead of a CAP-specific adaptation. However, CAP also affected bacterioferritin (Bfr), Isocitrate dehydrogenase (Idh), Trigger factor (Tig) and a chemotaxis protein, which may be involved in P. aeruginosa's specific response to CAP. We confirm that bacterioferritin B plays a role in the bacterial response to CAP because ΔbfrB mutants of both PAO1 and PA14 are more susceptible to plasma-induced cell-death than their corresponding wild-type strains. To our knowledge, this is the first study showing the effect of plasma on the whole proteome of a pathogenic microorganism. It will help our understanding of the mode of action of CAP-mediated bacterial inactivation and thus support a safe and effective routine use of CAP in clinical and industrial settings.