In recent decades, an enormous potential of fungal-based products with characteristics equal to, or even outperforming, classic petroleum-derived products has been acknowledged. The production of these new materials uses mycelium, a root-like structure of fungi consisting of a mass of branching, thread-like hyphae. Optimizing the production of mycelium-based materials and fungal growth under technical conditions needs to be further investigated. The main objective of this study was to select fast-growing fungi and identify optimized incubation conditions to obtain a dense mycelium mat in a short time. Further, the influence of the initial substrate characteristics on hyphae expansion was determined. Fungal isolates of
Ganoderma lucidum
,
Pleurotus ostreatus
, and
Trametes versicolor
were cultivated for seven days on substrate mixtures consisting of various proportions of pine bark and cotton fibers. Furthermore, the substrates were mixed with 0, 2, and 5 wt.% calcium carbonate (CaCO
3
), and the incubator was flushed with 0, 5, and 10 vol.% carbon dioxide (CO
2
). All samples grew in the dark at 26 °C and a relative humidity of 80%. Evaluation of growth rate shows that cotton fiber-rich substrates performed best for all investigated fungi. Although
Pleurotus ostreatus
and
Trametes versicolor
showed comparatively high growth rates of up to 5.4 and 5.3 mm d
−1
, respectively, mycelium density was thin and transparent.
Ganoderma lucidum
showed a significantly denser mycelium at a maximum growth rate of 3.3 mm d
−1
on a cotton fiber-rich substrate (75 wt.%) without CaCO
3
but flushed with 5 vol.% CO
2
during incubation.