The ribosomally synthesized and post-translationally modified peptide (RiPP) darobactin A is a promising new antibiotic candidate with anti-Gram-negative activity inflicted by the inhibition of the novel target BamA. Genome mining revealed many putative darobactin producer strains, but a limited number of compound modification options. In this study, the amber stop codon suppression technique was used to integrate non-canonical amino acids into the bicyclic heptapeptide, creating new darobactin derivatives. The C-terminal phenylalanine was replaced by non-canonical phenylalanine derivatives with different substituents. Darobactin A F7F, featuring a fluorine atom in the para position of the C-terminal phenylalanine, was purified to enable structure validation by NMR. Activity assays revealed antimicrobial potency against selected Gram-negative strains comparable to darobactin A.