Microbes have shown a remarkable ability in evading the killing actions of antimicrobial agents, such that treatment of bacterial infections represents once more an urgent global challenge. Understanding the initial bacterial response to antimicrobials may reveal intrinsic tolerance mechanisms to antibiotics and suggest alternative and less conventional therapeutic strategies. Here, we used mass spectrometry-based metabolomics to monitor the immediate metabolic response of Escherichia coli to a variety of antibiotic perturbations. We show that rapid metabolic changes can reflect drug mechanisms of action and reveal the active role of metabolism in mediating the first stress response to antimicrobials. We uncovered a role for ammonium imbalance in aggravating chloramphenicol toxicity and the essential function of deoxythymidine 5′-diphosphate (dTDP)-rhamnose synthesis for the immediate transcriptional upregulation of GyrA in response to quinolone antibiotics. Our results suggest bacterial metabolism as an attractive target to interfere with the early bacterial response to antibiotic treatments and reduce the probability for survival and eventual evolution of antibiotic resistance. Display omitted •Charting the metabolome response of E. coli to antibiotic treatment•Functional role of the rapid metabolic response in coping with antibiotic stress•The role for ammonium imbalance in aggravating chloramphenicol toxicity•dTDP-rhamnose regulates GyrA transcription in response to quinolone antibiotics Zampieri et al. monitor short-term metabolic changes in Escherichia coli after exposure to antibiotics. A core set of metabolites exhibits a unique rapid response to antibiotic with common or radically different modes of action. By interfering with such cellular response, the authors reveal the functional role of metabolism in mediating the first immediate response to antibiotics.