Antibiotic resistance is one of the most prominent threats to modern medicine. In the latest World Health Organisation (WHO) list of bacterial pathogens that urgently require the development of new antibiotics, nine out of 12 are Gram-negative, with four of those listed under Critical Priority. One important barrier restricting antibiotic efficacy against Gram-negative bacteria is their unique cell envelope. Fatty acids are a shared constituent of all structural membrane lipids and in bacteria, the Type II fatty acid synthesis pathway (FAS II) is a promising target for antibiotic development, as it is distinct from that of eukaryotes. However many FAS II components have evolved redundancy, and deemed unsuitable drug targets. Here, we interrogated the redundant components of FAS II, showing that disrupting FAS II homeostasis by deletion of fabH damages the cell envelope of clinical Escherichia coli isolates. The fabH gene encodes the β-ketoacyl acyl carrier protein synthase III (KAS III) which catalyses the initial condensation reactions during fatty acid biosynthesis. We found fabH null mutation potentiated killing of multi-drug resistant E. coli by a broad panel of previously ineffective antibiotics, despite the presence of relevant antibiotic resistance determinants (blaCTX-M-15 and kpc2). This sensitivity were transferrable to both biofilm eradication and the treatment of pre-established bladder cell monolayer infections. Our findings showcase the potential of FabH as a promising target that may rescue failing last-resort antibiotics.