Two methods of quantifying fungal activity have been compared and correlated: isothermal calorimetry for measuring heat production and gas chromatography–tandem mass spectrometry (GC–MS/MS) for measuring ergosterol, a proxy for biomass. The measurements were made on four different fungi: Penicillium roqueforti, Cladosporium cladosporioides, Neopetromyces muricatus and the dry rot fungus Serpula lacrymans. The results showed linear correlations between ergosterol production and total heat production for these four fungal species during the initial fast growing stage. At the later stages heat was produced but ergosterol amount was constant. The heat produced per ergosterol amount varied from species to species and between different temperatures. This might be due to the different metabolic efficiencies of different species or the same species at different temperatures. Isothermal calorimetry can be used in fungal studies on its own or in combination with other techniques for a more complete understanding of fungal physiology.