<jats:title>Abstract</jats:title><jats:p>This work aimed to undertake the in situ conversion of the terminal amine groups of bacterial desferrioxamine (DFO) siderophores, including desferrioxamine B (DFOB), to azide groups to enable downstream click chemistry. Initial studies trialed a precursor‐directed biosynthesis (PDB) approach. Supplementing <jats:italic>Streptomyces pilosus</jats:italic> culture with blunt‐end azido/amine non‐native substrates designed to replace 1,5‐diaminopentane as the native diamine substrate in the terminal amine position of DFOB did not produce azido‐DFOB. Addition of the diazo‐transfer reagent imidazole‐1‐sulfonyl azide hydrogen sulfate to spent <jats:italic>S. pilosus</jats:italic> medium that had been cultured in the presence of 1,4‐diaminobutane, as a viable native substrate to expand the suite of native DFO‐type siderophores, successfully generated the cognate suite of azido‐DFO analogues. Cu<jats:sup>I</jats:sup>‐mediated or strain‐promoted Cu<jats:sup>I</jats:sup>‐free click chemistry reactions between this minimally processed mixture and the appropriate alkyne‐bearing biotin reagents produced the cognate suite of 1,4‐disubstituted triazole‐linked DFO‐biotin compounds as potential molecular probes, detected as Fe<jats:sup>III</jats:sup>‐loaded species. The amine‐to‐azide transformation of amine‐bearing natural products in complex mixtures by the direct addition of a diazo‐transfer reagent to deliver functional click chemistry reagents adds to the toolbox for chemical proteomics, chemical biology, and drug discovery.