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The multiple forms of pulmonary aspergillosis caused by Aspergillus species are the most common respiratory mycoses. Although invasive, the analysis of diagnostic biomarkers in bronchoalveolar lavage fluid (BALF) is a clinical standard for diagnosing these conditions. The BALF samples from 22 patients with proven or probable aspergillosis were assayed for human pentraxin 3 (Ptx3), fungal ferricrocin (Fc), and triacetylfusarinine C (TafC) in a retrospective study. The infected group included patients with invasive pulmonary aspergillosis (IPA) and chronic aspergillosis (CPA). The BALF data were compared to a control cohort of 67 patients with invasive pulmonary mucormycosis (IPM), non-Aspergillus colonization, or bacterial infections. The median Ptx3 concentrations in patients with and without aspergillosis were 4545.5 and 242.0 pg/mL, respectively (95% CI, p < 0.05). The optimum Ptx3 cutoff for IPA was 2545 pg/mL, giving a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 100, 98, 95, and 100%, respectively. The median Ptx3 concentration for IPM was high at 4326 pg/mL. Pentraxin 3 assay alone can distinguish IPA from CPA and invasive fungal disease from colonization. Combining Ptx3 and TafC assays enabled the diagnostic discrimination of IPM and IPA, giving a specificity and PPV of 100%.