Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that produces highly antibiotic-tolerant biofilms that create an enormous therapeutic burden as they cause many types of chronic infections. The reason P. aeruginosa biofilms are problematic is in part due to reduced growth activity and downregulation of metabolic processes within biofilms that are typically the target of antibiotics. There is an unmet need for novel strategies that aim to either disrupt the biofilm itself or modulate metabolic activity and cell survical mechanisms within biofilms. A relatively unexplored mechanism for disrupting the metabolic adaptability of P. aeruginosa is via inhibition of serine/threonine phosphorylation pathways of eukaryote-like serine/threonine kinases in biofilms. These kinases are characterised by a catalytic Hanks-type domain that reversibly phosphorylates serine or threonine residues of various proteins in many cellular processes, including several regulatory proteins of other signalling cascades. The focus of this study was to investigate the role of eSTK’s in P. aeruginosa biofilm development and their impact on metabolism and adaptive responses. There are four eSTK’s in the P. aeruginosa genome. All four of these genes were deleted from PAO1 individually and sequentially to produce a quadruple knockout mutant termed QKO. In addition, three were deleted individually from P. aeruginosa PA2192, a cyctic fibrosis clinical isolate. Using multiple strategies such as confocal microscopy and TMT-labelled proteomics analysis we showed that deletion of a single eSTK, YeaG, of PA2192 can inhibit aggregate formation, and that the metabolic adaptability of the QKO mutant of PAO1 is significantly disrupted and which leads to major alterations within intracellular signalling pathways. This leads to a compromised membrane phenotype that allows greater influx of positively charged compounds such as propidium iodide. Although we did not see any increase in the susceptibility of the QKO mutant to tobramycin in the model used, the apparent cellular stress exhibited by the QKO mutant and the inhibited aggregation of the PA2192 suggest that eSTK’s are potentially interesting drug targets with therapeutic potential in the control of P. aeruginosa biofilms.