<jats:title>Abstract</jats:title><jats:p>Polymyxins are last-resort antibiotics for treating infections of Gram-negative bacteria. The recent emergence of polymyxin-resistant bacteria, however, urgently demands clinical optimisation of polymyxin use to minimise further evolution of resistance. In this study we developed a novel combination therapy using minimal concentrations of polymyxin B. After large-scale screening of <jats:italic>Streptomyces</jats:italic> secondary metabolites, we identified a reliable polymixin synergist and confirmed as netropsin using high-pressure liquid chromatography, nuclear magnetic resonance and mass spectrometry followed by <jats:italic>in vitro</jats:italic> assays using various Gram-negative pathogenic bacteria. To evaluate the effectiveness of combining polymixin B and netropsin <jats:italic>in vivo</jats:italic>, we performed survival analysis on greater wax moth <jats:italic>Galleria mellonella</jats:italic> infected with colistin-resistant clinical <jats:italic>Acinetobacter baumannii</jats:italic> isolates as well as <jats:italic>Escherichia coli, Shigella flexineri, Salmonella typhimuruim,</jats:italic> and <jats:italic>Pseudomonas aeruginosa</jats:italic>. The survival of infected <jats:italic>G. mellonella</jats:italic> was significantly higher when treated with polymyxin B and netropsin in combination than when treated with polymyxin B or netropsin alone. We propose a netropsin combination therapy that minimises the use of polymyxin B when treating infections with multidrug resistant Gram-negative bacteria.