Bioconversion of plant material with white-rot fungi has demonstrated improvements in in vitro digestibility, however, effects on rumen fermentation parameters and methane (CH4) production, when included as a dietary ingredient, are not clear. The aim of this study was to assess changes in rumen fermentation parameters and in vitro CH4 production by dietary inclusion of ryegrass-fescue hay biodegraded with P. ostreatus (H-PO) during 14 days of solid fermentation. The experiment used 2 diets with contrasting acid detergent fibre (aADFom) content which were added with 0, 3 and 6% H-PO. Substrates were incubated 3 times with sampling at 24 h. Results were analysed by ANOVA, and compared with Tukey's test and polynomial contrasts. Inclusion of H-PO at concentrations of 3 and 6% resulted in increased in vitro dry matter (DM) disappearance (IVDMD) by 10 and 12% respectively, whereas high concentrations of aADFom reduced IVDMD by 10% as compared to low aADFom. Methane production (mg/g DM incubated) and yield (mg/g DM degraded) were reduced by increasing aADFom and P. ostreatus biodegraded hay concentrations in the diets (p < 0.001) whereas concentrations of total short-chain fatty acids were not affected by the inclusion of biodegraded hay (p = 0.080) or aADFom concentration (p = 0.076). Increasing concentrations of biodegraded hay resulted in increased acetate (p < 0.001), and reduced propionate proportions (p = 0.021) but did not affect butyrate (p = 0.233). In conclusion the use of biodegraded hay as a pre-treated ingredient improved the utilisation of low and high fibre diets by increasing the in vitro disappearance, and decreasing CH4 emissions, without increasing the propionate proportion.HIGHLIGHTSThe use of hay biodegraded with P. ostreatus (H-PO) improves the digestibility of ruminant diets.The use of H-PO decreases methane, and increases acetate production in in vitro conditions.The use of H-PO as an ingredient in ruminant diets could improve the sustainability of livestock systems.