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The aim of the study was to evaluate the influence of hypoxia on galactomannan and (1,3)-&#946;-<span style="font-variant: small-caps;">d</span>-glucan release of clinically relevant <i>Aspergilli</i> in vitro. Hypoxia decreased biomass and consequently led to lower biomarker release. However, when normalized to biomass, hypoxia led to increased levels of biomarkers at early growth stages (24 h). Antifungals (amphotericin B and voriconazole) decreased the galactomannan amount of <i>A. fumigatus</i>, even more prominently in hypoxia.