A mass spectrometry (MS) method that detects a serum disaccharide (DS) (MS-DS) was recently described for the diagnosis of invasive fungal infections (IFI). We carried out a European collaborative study to evaluate this assay. Patients with the following IFI were selected according to the availability of sera obtained at about the time that IFI was documented: invasive candidiasis (IC; n = 26 patients), invasive aspergillosis (IA; n = 19), and mucormycosis (MM; n = 23). Control sera originated from 20 neutropenic patients and 20 patients with bacteremia. MS-DS was carried out in blind manner for the diagnosis of IFI. A diagnosis of IC or IA was confirmed by detection of mannan (Man) or galactomannan (GM), respectively, associated with detection of (1,3)-β-d-glucan (BDG) in both infections. MM was detected by quantitative real-time PCR (qPCR). All tests discriminated sera from patients with IC from sera from control subjects with bacteremia (P ≤ 0.0009). For IC, the MS-DS sensitivity and specificity were 51% and 87%, respectively. MS-DS complemented the high specificity of Man monitoring. All tests discriminated sera from IA patients from sera from neutropenic controls (P ≤ 0.0009). For IA, MS-DS sensitivity and specificity were 64% and 95%, respectively. Only 13/36 serum samples from patients with MM were concordant by MS-DS and qPCR (6 were positive, and 7 were negative); 14 were positive by MS-DS alone. qPCR and MS-DS made a similar contribution to the diagnosis of MM. In patients undergoing long-term monitoring, the persistent circulation of serum disaccharide was observed, whereas DNA was detected only for a short period after initiation of treatment. MS-DS has an important role to play in the early diagnosis of IFI. Its panfungal nature and complementarity with other tests may justify its use in the management of IFI.