Lysine acetylation (KAc) is a reversible and dynamic post-translational modification (PTM) that regulates a myriad of fundamental biological processes in bacteria, including metabolism and processes related to virulence. KAc can occur either enzymatically via protein acetyltransferase (KAT) activity; or chemically, which is governed by intracellular levels of the metabolic intermediate, acetyl-phosphate (AcP). Removal of the acetyl group is mediated by virtue of the major sitruin-like deacetylase ortholog, CobB. While global acetylome studies have identified sites of KAc in various bacteria, the acetylome of the pathogen P. aeruginosa remains poorly characterized. Additionally, unlike all other known bacteria, P. aeruginosa contains two copies of cobB (cobB1 and cobB2) and no studies have determined their targets. Here, we conducted a global proteomic analysis of KAc in P. aeruginosa mPAO1 with combined acetyl-lysine immunoprecipitation and label-free liquid chromatography tandem mass spectrometry. Label-based LC–MS/MS was used to quantify relative differences in peptide and acetylated peptide abundances between the wild-type (WT) parent mPAO1 and ΔcobB1/ΔcobB2 deletion mutants. Complementary plate-based assays assessed motility and biofilm formation of the ΔcobB1/B2 strains. Comparative analysis identified 3425 proteins, with those involved in chemotaxis altered in one, or both, mutants. Phenotypic assays show increased swimming motility in ΔcobB P. aeruginosa, while biofilm formation was attenuated compared to WT. We have defined the global acetylome in P. aeruginosa and identified shared and specific CobB1/B2 sites. These results provide significant insights into the role of KAc in P. aeruginosa.