The Fungitell assay (FA) and the Wako β-glucan test (GT) are employed to measure the serum/plasma 1,3-β-D-glucan (BDG), a well-known invasive fungal disease biomarker. Data to convincingly and/or sufficiently support the GT as a valuable alternative to the FA are yet limited. In this study, we evaluated the FA and the GT to diagnose invasive aspergillosis (IA), invasive candidiasis (IC), and Pneumocystis jirovecii pneumonia (PJP). The FA and GT performances were compared in sera of patients with IA (n = 40), IC (n = 78), and PJP (n = 17) with respect to sera of control patients (n = 187). Using the manufacturer's cutoff values of 80 pg/mL and 11 pg/mL, the sensitivity and specificity for IA diagnosis were 92.5% and 99.5% for the FA and 60.0% and 99.5% for the GT, respectively; for IC diagnosis were 100.0% and 97.3% for the FA and 91.0% and 99.5% for the GT, respectively; for PJP diagnosis were 100.0% and 97.3% for the FA and 88.2% and 99.5% for the GT, respectively. When an optimized cutoff value of 7.0 pg/mL for the GT was used, the sensitivity and specificity were 80.0% and 97.3% for IA diagnosis, 98.7% and 97.3% for IC diagnosis, and 94.1% and 97.3% for PJP diagnosis, respectively. At the 7.0-pg/mL GT cutoff, the agreement between the assays remained and/or became excellent for IA (95.1%), IC (97.3%), and PJP (96.5%), respectively. In conclusion, we show that the GT performed as well as the FA only with a lowered cutoff value for positivity. Further studies are expected to establish the equivalence of the two BDG assays.