Antimicrobial resistance has become a significant threat to human health and it demands urgent action to develop new therapeutic agents with novel mechanisms of action.1 Recently, a new cationic non-ribosomal peptide (CNRP), brevicidine, was discovered from Brevibacillus laterosporus through a global-genome mining approach.2 Brevicidine exhibits potent antimicrobial activity toward Gram-negative bacteria, including colistin-resistant Escherichia coli, and a low risk of resistance development. Herein, we report the first total synthesis of brevicidine via an efficient and high-yielding synthetic pathway, employing both solid-phase synthesis and solution-phase macrolactamization. The crucial depsipeptide bond of the macrolactone ring of brevicidine was established on-resin between the side-chain hydroxy group of Thr9 with Alloc-Ser(tBu)-OH. A conserved glycine residue within the lactone macrocycle was exploited for the initial immobilisation onto the hyper acid-labile 2-chlorotrityl chloride resin, subsequently enabling an efficient solution-phase macrocyclization to yield brevicidine in 10% overall yield (with respect to resin loading). A biological evaluation against both Gram-positive and Gram-negative bacteria demonstrated that synthetic brevicidine possessed a potent and selective antimicrobial activity toward Gram-negative bacteria, in agreement with the isolated compound.