Polymyxins are amongst the most important antibiotics in modern medicine, in recent times their clinical utility has been overshadowed by nosocomial outbreaks of polymyxin resistant MDR Gram-negative 'superbugs'. An effective strategy to surmount polymyxin resistance is combination therapy with FDA-approved non-antibiotic drugs. Herein we used untargeted metabolomics to investigate the mechanism(s) of synergy between polymyxin B and the selective estrogen receptor modulator (SERM) tamoxifen against a polymyxin-resistant MDR cystic fibrosis (CF) Pseudomonas aeruginosa FADDI-PA006 isolate (polymyxin B MIC=8 mg/L , it is an MDR polymyxin resistant P. aeruginosa isolated from the lungs of a CF patient). The metabolome of FADDI-PA006 was profiled at 15 min, 1 and 4 h following treatment with polymyxin B (2 mg/L), tamoxifen (8 mg/L) either as monotherapy or in combination. At 15 min, the combination treatment induced a marked decrease in lipids, primarily fatty acid and glycerophospholipid metabolites that are involved in the biosynthesis of bacterial membranes. In line with the polymyxin-resistant status of this strain, at 1 h, both polymyxin B and tamoxifen monotherapies produced little effect on bacterial metabolism. In contrast to the combination which induced extensive reduction (≥ 1.0-log2-fold, p ≤ 0.05; FDR ≤ 0.05) in the levels of essential intermediates involved in cell envelope biosynthesis. Overall, these novel findings demonstrate that the primary mechanisms underlying the synergistic bactericidal effect of the combination against the polymyxin-resistant P. aeruginosa CF isolate FADDI-PA006 involves a disruption of the cell envelope biogenesis and an inhibition of aminoarabinose LPS modifications that confer polymyxin resistance.