Invasive aspergillosis is an important cause of morbidity and mortality in immunocompromised patients. Diagnosis of this infection frequently employs detection of the circulating galactomannan in the patient serum using enzyme immunoassay (EIA), a highly sensitive and specific system. Although there are many structural studies of the galactomannan of Aspergillus fumigatus, some inconsistencies are present in these results. In this study, to clarify the relationship between the growth conditions and structure of the galactomannans, we cultured A. fumigatus using two distinct yeast/fungal cultivation media, i.e. the yeast extract–peptone–dextrose (YPD) medium and yeast nitrogen base (YNB) medium. Galactomannans prepared from the resulting culture supernatants were structurally characterized by 1H and 13C nuclear magnetic resonance, methylation analysis, acetolysis and α-mannosidase degradation. These assays revealed that the galactomannan from the YPD cultivation had short β-1,5-linked galactofuranose (Galf) oligosaccharide chains in both the O- and N-linked carbohydrate moieties, while the galactomannan from the YNB cultivation incorporated long Galf oligosaccharide chains. The galactomannans derived from the two culture conditions significantly differed in reactivity based on the EIA diagnostic system. We also demonstrated the presence of a novel Galf-containing branched oligosaccharide in the O-linked moiety.