Biological treatment of cereal straw for ruminant nutrition purposes might present an environmentally friendly option of valorizing a widely available by-product of grain production for farming systems with low external input. Several strains of white-rot fungi have been selected in the past under mostly controlled laboratory conditions for their capacity of lignin degradation. The study adapted to conditions on farm for upscaling purposes. The development of the in vitro straw digestibility with two different moistening pre-treatments and inoculated with three different fungi species, namelyPleurotus ostreatus,Ceriporiopsis subvermisporaandVolvariella volvacea, was determined up to 42 days of fermentation with five sampling times. The effect of physical straw pre-treatments on nutritional parameters was evaluated. The neutral detergent fiber digestibility (NDFD30h), enzymatically soluble organic substance (ELOS) and the gas production (Hohenheim Feed value Test, HFT) as indicators for in vitro ruminal degradability decreased over time independent of the fungus: HFT, ELOS and NDFD30hby up to 50, 35 and 30% of the original straw. Remoistening and autoclaving the straw increased the gas production significantly by 2.6 mL/200 g dry matter (DM), and ELOS and NDFD30hby 45 and 51 g/kg DM compared to the original straw (34.9 mL/200 mg DM, 342 g/kg DM, 313 g/kg NDF).