Acinetobactin is a major siderophore utilized by the human pathogen Acinetobacter baumannii. The rapid acquisition of drug resistance by A. baumannii has garnered concern globally. Herein, acinetobactin and systematically generated analogues were prepared and characterized; the binding and cellular delivery of Fe(III) by the analogues were evaluated. This investigation not only led to the clarification of the physiologically relevant acinetobactin structure but also revealed several key structural elements for its functionality as a siderophore.