Hydrogen peroxide is suspected of being highly implicated in mushroom nutrition and in substrate bleaching during cultivation. The parameters for measuring H2O2 in compost samples were examined and the methodology was applied to samples from both compost colonized by cultivars and wild isolates of Agaricus bisporus, and wheat straw or coffee pulp colonized by Pleurotus spp. Laccase and peroxidase activities were also measured. H2O2 concentration measured after heating at 80 °C for inactivating laccases and peroxidases was probably both H2O2 pre-existing in the compost and H2O2 generated from quinones and active oxygen species. This potential H2O2 concentration increased during the vegetative growth for all the strains, in agreement with a direct relationship between H2O2 concentration and active biomass of A. bisporus or Pleurotus spp. in their cultivation substrates. Correlations were observed between H2O2 concentration and manganese peroxidase activity in cultivation substrates at the stage of primordia formation. At this stage of development, H2O2 generation via biotic or abiotic mechanisms should be an important physiological trait of mushrooms.