Polymyxins are naturally occurring cyclic lipopeptides that were discovered more than 60Â years ago. They have a narrow antibacterial spectrum, which is mainly against Gram-negative pathogens. The dry antibiotic pipeline, together with the increasing incidence of bacterial resistance in the clinic, has been dubbed â€˜the perfect stormâ€™. This has forced a re-evaluation of â€˜oldâ€™ antibiotics, in particular the polymyxins, which retain activity against many multidrug-resistant (MDR) Gram-negative organisms. As a consequence, polymyxin B and colistin (polymyxin E) are now used as the last therapeutic option for infections caused by â€˜superbugsâ€™ such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. This chapter covers the history, chemistry and antibacterial spectrum of these very important last-line lipopeptide antibiotics.