A colorimetric method was developed to permit semi-quantitative measurement of substrate acidification by different ectomycorrhizal and one saprotrophic fungus growing on media containing one of five different minerals. Overall, substrate acidification differed between fungal species and the degree of variation in acidification in response to different minerals was highly species-dependent. Mycena galopus and Cortinarius glaucopus produced the least biomass of all tested species and produced the highest amount of acidification per unit mycelial density. Substrate acidification by C. glaucopus was inversely related to mycelial density, with particularly high acidification at low mycelial density on medium enriched with tri-calcium phosphate. Substrate acidification by M. galopus was constant irrespective of mycelial density and varied only according to mineral treatment, with higher substrate acidification on tri-calcium phosphate compared to the other minerals.