Antimicrobial resistance (AMR) is a major global problem and new ways of identifying antibiotic targets are essential. Gliotoxin is a potent antimicrobial with broad spectrum activity produced by Aspergillus fumigatus. The toxicity of gliotoxin was previously attributed to reactive oxygen species (ROS) production resulting from conversion between the oxidised (GT) and reduced (DTG) forms. However, the nascent zinc-chelating ability of DTG inspired research into a metallo-centric model of bacterial growth inhibition. The ROS model has been augmented by work herein showing gliotoxin is a broad-spectrum antimicrobial whose toxicity is attenuated by specific metal ion supplementation (zinc, copper). This finding was supported by unbiased LFQ proteomics revealing gliotoxin induced perturbation of metal, primarily zinc, homeostasis systems in both Gram-positive and -negative pathogens, including Adc, Znu, and TonB metal uptake proteins, ribosomal remodelling to zinc free paralogs, and the activation of the Fur controlled acinetobactin biosynthesis gene cluster. Pre-formed DTG chelates caused growth inhibition, and promotion in the case of DTG:Cu, positing DTG as a potential ionophore. This metallo-centric model was supported by experiments showing DTG ejects zinc, copper, and iron from chromogens (PAR, Siderotec™ Total, Siderotec™ HiSens). DTG also ejected zinc from protein extracts in vitro. Despite this, DTG did not cause intracellular zinc depletion. This multi-metal chelating ability was confirmed using mass spectrometric analysis to detect DTG:Zn, DTG:Cu, and DTG2:Fe chelates. Thioacetylated DTG was synthesised which requires future study but may be usable as an attenuated form of gliotoxin which can be administered alone or in combination with antibiotics. Co-addition studies using gliotoxin and vancomycin revealed a zinc dependent additive effect. Proteomic analysis revealed significant alterations due to combination treatment and perturbation of several key systems including cell wall biosynthesis and several transcriptional regulators. Overall, this work provides new insight into the Pathfinder role of gliotoxin in the fight against AMR.