Degradation of lignocellulose by selective white-rot fungi can significantly improve the nutritional value of high lignocellulose containing biomass by affecting the access of rumen microbes to structural carbohydrates. To determine if such treated biomass is stable in time to allow it to be conserved for subsequent feeding to ruminant animals, wheat straw (WS) pre-treated for 7 weeks with either Ceriporiopsis subvermispora or Lentinula edodes was stored anaerobically up to 10 weeks at different temperatures (24.7–52.4 °C). Substrates were subsequently analysed for changes in pH, titratable acidity, fibre composition, in vitro gas production (IVGP) and colour, as well as polar metabolites by GC–MS and ceriporic acids by LC–MS. The increased titratable acidity of fungal treated WS during storage indicated acidification of the straw. A significant decrease in hemicellulose and an increase in acid detergent lignin content was observed at 52.4 °C. No negative effect of the storage condition on the degradability of both fungal treated WS in rumen fluid was observed. A darker colour was observed for substrates incubated at higher temperatures, coinciding with a strong accumulation of several organic acids and sugars. A decrease in ceriporic acid A, B, C and G produced by C. subvermispora was observed when stored at 52.4 °C from week 2–10. The results show that, although the chemical composition changes, anaerobic storage of fungal treated WS at different temperatures does not affect its fermentation potential for ruminants.