The aim of this experiment was to determine the effect of spent mushroom substrate and mineral nitrogen and potassium fertilisation on the yield of dry matter and protein of orchard grass (Dactylis glomerata L.). The three-year field experiment in the completely randomised design was conducted on a soil with the particle size distribution of light loamy sand and with the use of organic materials which were cattle farmyard manure and substrate after the cultivation of mushrooms. The spent mushroom substrate used in the experiment was richer in dry matter and total nitrogen, and had lower content of organic carbon, total phosphorus and potassium in relation to farmyard manure. The greatest total yield of biomass and protein of orchard grass in each study year  was recorded in the treatments with mushroom substrate and a higher dose of mineral N and K.