Production of carbon dioxide, as one of the ultimate products of fungal metabolism, can be used to quantify and measure their metabolic rate under different conditions, thus aiding in finding the optimal substrate and environment for cultivation of wood-destroying fungi. This study is focused on species Pleurotus ostreatus and Ganoderma lucidum,. These species are also cultivated for mycorestoration as well as their medicinal and nutritional value. To quantify their metabolical rate on various substrates (agar medium, wood chips, rye straw), multiple custom-built airtight chambers were equipped with CO2 probes (GMP 343, Vaisala, Finland) to measure the production of carbon dioxide. The highest values were measured during the primordial production on rye straw substrate, with the average values of 1.09 g CO2 kg-1 (substrate) h-1. These values varied significantly between various substrates, fungal species and development stages.