Siderophores are a structurally diverse class of natural products common to most bacteria and fungi as iron(III)-chelating ligands. Siderophores， including trihydroxamate ferrioxamines， are used clinically to treat iron overload diseases and show promising activity against many other iron-related human diseases. Here， we present a new method for the isolation of ferrioxamine siderophores from complex mixtures using affinity chromatography based on resin-immobilized FhuD2， a siderophore-binding protein (SBP) from Staphylococcus aureus. The SBP-resin enabled purification of charge positive， charge negative， and neutral ferrioxamine siderophores. Treatment of culture supernatants from Streptomyces violaceus DSM 8286 with SBP-resin provided an analytically pure sample of the salmycins， a mixture of structurally complex glycosylated sideromycins (siderophore-antibiotic conjugates) with potent antibacterial activity toward human pathogenic Staphylococcus aureus (minimum inhibitory concentration (MIC) = 7 nM). Siderophore affinity chromatography could enable the rapid discovery of new siderophore and sideromycin natural products from complex mixtures to aid drug discovery and metabolite identification efforts in a broad range of therapeutic areas.