The brevicidines represent a novel class of non‑ribosomal antimicrobial peptides that possess remarkable potency and selectivity towards highly problematic and resistant Gram-negative pathogenic bacteria. A recently discovered member of the brevicidine family, coined brevicidine B, is reported to contain a single amino acid substitution (from D-Tyr² to D-Phe²) in the amino acid sequence of the linear moiety of brevicidine and has an extended antimicrobial spectrum toward both Gram‑negative (MIC = 2‑4 μgmL^-1) and Gram‑positive (MIC = 2-8 μgmL^-1) pathogens. Encouraged by this, we successfully undertook the first total synthesis of brevicidine B, building on the synthetic strategy we previously reported to access brevicidine. In agreement with the original report, synthetic brevicidine B demonstrated potent antimicrobial activity toward Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae (MIC = 4-8 μgmL^-1). Interestingly however, synthetic brevicidine B proved inactive towards all of the tested Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus strains. Substitution of D‑Phe² with its enantiomer, and other hydrophobic residues, yields analogues that were either inactive or exhibited activity only towards Gram‑negative strains. The striking difference in the biological activity of our synthetic brevicidine B compared to the reported compound warrants the re-evaluation of the original natural product for purity or existence of stereochemical differences. Finally, evaluation of synthetic brevicidine and brevicidine B in a human kidney organoid model of nephrotoxicity revealed substantial toxicity of both compounds, although brevicidine was less toxic brevicidine B and polymyxin B.