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Summary: Background: Invasive pulmonary aspergillosis (IPA) remains a life‐threatening opportunistic infection, but can be difficult to diagnose. New biomarkers are therefore needed. Gliotoxin (GT), a secondary metabolite of Aspergillus fumigatus, and bis(methylthio)gliotoxin (bmGT), a degradation product of GT, have been proposed as potential biomarkers. However, these findings have yet to be confirmed. Objectives: To identify the diagnostic potential of GT and bmGT in serum and bronchoalveolar lavage fluid (BALf) in haematology patients compared to galactomannan (GM). Materials and methods: We prospectively collected culture supernatant, serum and BALf from patients with culture‐positive IPA and measured GT and bmGT concentrations using ultra high‐performance liquid chromatography–quadrupole time of flight mass spectrometry. Galactomannan was detected using a commercially available enzyme immunoassay. Results: We included 18 patients with proven (n = 6) and probable (n = 12) IPA, all with positive cultures for Aspergillus fumigatus. BmGT was only detected in serum from one patient (5.6%), whereas GM was positive (optical density ≥ 0.5) in 11/18 patients (61.1%, P = 0.002). We could not find GT in any serum sample. In BALf, bmGT was detected in 8/18 patients (44.4%) and GT in 9/18 patients (50%), compared to GM (optical density ≥ 1.0) in all patients (100%). Conclusions: Gliotoxin and bis(methylthio)gliotoxin had a very poor performance for diagnosing IPA. As other biomarkers are more sensitive and easier to detect, we would not recommend serum or BALf GT/bmGT to be used in the diagnosis of IPA. [ABSTRACT FROM AUTHOR]