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 (CaCO3), and the incubator was flushed with 0, 5, and 10 vol.% carbon dioxide (CO2). 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 CaCO3 but flushed with 5 vol.% CO2 during incubation.