Hydroxamate siderophores are low molecular weight, high-affinity ferric iron-chelating compounds which are excreted by Aspergillus fumigatus for iron transport extracellularly or iron storage intracellularly. Aspergillus fumigatus was isolated from different samples, then siderophores were extracted from A. fumigatus by phenol, and diethyl ether, and purified by amberlite XAD-16 resin. The chemical structure of siderophores was described by H¹NMR,ESI-MS/MS, andLC-MS/MS analysis. Also, antimicrobial activity was observed. From the elution profile of siderophores, the major peak was at fraction 28.  As a result of H¹NMR analysis, the singlets assigned to the -C=O-CH=C (CH3) C-substructure were detected. The siderophore TafC had antibacterial activity against both G +ve and G -ve bacteria. Under different concentrations of iron, it can be deduced that the concentration of 0.03 mmol of iron at 20 hrs. incubation period was an ideal condition for mycelial fresh weight which positively affected, so production of siderophores increased. From chemical analysis, it was found that the chemical formula C₃₉H₅₈FeN₆O₁₅ was consistent with the known siderophore N, N', N"-triacetylfusarinine C (TafC).