New lipidation approaches are of increasing interest in the field of peptide chemistry. A chemoselective S-lipidation strategy in comparison with conventional acylation, not only offers a more efficient route for late-stage structural derivatisation of a peptide scaffold, but can also provide access to chemically diverse compounds possessing intriguing and non-native moieties. Herein, we report the first semi-synthesis of antimicrobial lipopeptides, leveraging a highly efficient thia-Michael addition with a chemically diverse range of lipophilic thiols. Taking chemoenzymatically prepared polymyxin B nonapeptide (PMBN) as a model scaffold, an allenamide mediated thia-Michael addition was optimised to provide rapid and near quantitative lipidation, yielding vinyl sulfide-linked polymyxin lipopeptide derivatives in up to 25 % yield over five synthetic steps. With the utility of this new methodology, lipophilic thiols of unprecedented chemical diversity were introduced to the PMBN framework, including heterocyclic thiols and those bearing additional functional groups (e.g. amines), ultimately yielding analogues with potent Gram-negative antimicrobial activity and substantially attenuated nephrotoxicity.