Zinc is central to the function of many proteins, yet the mechanisms of zinc homeostasis and its impact on other cellular regulatory systems remain under-characterized. In this study, we used the opportunistic human pathogen and environmentally significant model organism Pseudomonas aeruginosa to explore zinc activity via proteomic methods. Overall, we identified 2120 proteins using data-dependent acquisition mass spectrometry and 1909 proteins using data-independent acquisition mass spectrometry, 1667 of which were shared between the two methods. We showed that ATP-binding cassette (ABC) transporters are significant methods of zinc uptake in this system via upregulation of associated proteins when P. aeruginosa was grown under low zinc conditions. Previously identified zinc efflux mechanisms, such as P-type ATPase transporter ZntA and resistance-nodulation-cell division cation efflux transporter CzcA responded to high zinc conditions, emphasizing the importance of tight regulation of intracellular zinc in both depleted and replete habitats. Protein associated with biofilm formation was impacted by the presence of zinc and the ability of the strains used to produce pseudopaline, a biogenic zinc-complexing molecule. Pseudopaline-deficient strains, which lacked the CntM synthesis protein, contained more abundant protein biomarkers for the formation of mature biofilms. We speculate that a previously undescribed system exists in which pseudopaline complexation of extracellular zinc provides a protection from free zinc ions. In the absence of this compound, zinc ions induce a RsmA-GacS/A mediated response that promotes the transition to a biofilm lifestyle.