Barley (Hordeum vulgare L.), and its derivatives, ranks fourth in cereal production worldwide, and the Pleurotus species are among the most efficient types of lignocellulolytic white-rot fungi. The objective of this research study was to evaluate the degradation of barley straw and barley rootless with an inoculum of Pleurotus to improve their nutritional availability as a food source for ruminants. Two experiments were conducted; the first was to determine the effects of inoculation of Pleurotus sapidus (Schulzer) Sacc. (PS) in barley straw (BS), barley rootless (BR), and a 75% BS and 25% BR mixture (M). The second experiment was to evaluate the same substrates in vitro ruminal fermentation. Barley rootless had better organic matter (OM) degradability than BS after 24 h incubation with PS. The protein content in BR was higher than in BS (P < 0.01). Enzyme activities had the highest concentration from the start of fermentation, and in vitro dry matter (DM) degradability in BS and BR increased after 8 and 24 d fermentation, respectively (P < 0.05). Propionic acid concentration was enhanced after 16 d fermentation in BR (P < 0.5). The use of BS combined with BR exhibited better fermentation; this result provides relevant information for integrating BR with other substrates and improving the use of straw, which can be more nutritionally available for feeding ruminants.