The aim of the research was to assess the effect of mushroom substrate and slurry on the content of structural and non-structural carbohydrates in hybrid alfalfa mixtures with grasses. The three-year research was conducted in an experimental field between 2013 and 2015, with the following variables: (1) spent mushroom substrate (SMS) and cow slurry (CS), applied in different combinations; (2) three legume grass mixtures: orchard grass, perennial ryegrass, hybrid alfalfa (M1); orchard grass, hybrid alfalfa (M2); perennial ryegrass, hybrid alfalfa (M3). In each growing season, the mixtures were harvested three times. Plant material was used to determine dry matter content and the content of neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL), cellulose, hemicellulose, lignin, total protein, crude ash, and crude fat by near infrared spectroscopy (NIRS), using the NIRFlex N-500 spectrometer and ready-to-use INGOT® calibration applications. Generally, mushroom substrate applied on its own increased the amounts of cellulose and hemicellulose in legume grass mixtures more than slurry. The most cellulose, the least hemicelluloses, and the highest degree of lignification were recorded in the mixture of ryegrass with alfalfa, while the degree of lignification was the smallest in the forage of alfalfa and orchard grass, which also contained the least cellulose but the most hemicellulose. On average, the highest amount of cellulose was in the biomass of the first harvest and the least in the third.