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The aim of this paper is to evaluate the effect of different doses of spent mushroom substrate and cow slurry on sugar content and digestibility of hybrid alfalfa and grass mixtures. The main factors were different doses of organic material: mushroom substrate and slurry, and the following legume grass mixtures: M1-orchard grass (Dactylis glomerata), perennial ryegrass (Lolium perenne), and hybrid alfalfa (Medicago x varia T. Martyn); M2-orchard grass, hybrid alfalfa; M3-perennial ryegrass, hybrid alfalfa. In each growing season, the mixtures were harvested three times during three years of their full use. Sugar content and dry matter digestibility were determined with near-infrared spectroscopy (NIRS) using the NIRFlex N-500 spectrometer. Of all fertilizer treatments, the application of mushroom substrate at a dose of 20 t·ha−1 in combination with 40 m3 of slurry resulted in the best forage quality with its highest digestibility. In the mixture of perennial ryegrass and hybrid alfalfa increasing doses of mushroom substrate with decreasing doses of slurry lowered soluble sugar content and digestibility. [ABSTRACT FROM AUTHOR]