This work presents the analysis of the effect of grassland management on leaching of mineral nitrogen from rhizosphere and plant biomass production in area of our interest. The determined values show the influence of fertilization and species composition of permanent grassland on leaching of mineral nitrogen and biomass production. The values were determined during field and laboratory experiments. The highest availability of Nmin in the field experiment was detected in the variant N90+PK; the lowest availability of Nmin was recorded in the PK variant. The difference was statistically significant (P < 0.05). In the pot experiment, the highest loss of Nmin was recorded in the variant with N180+PK (16.88 mg.dm−3) with the difference being statistically significant as compared with the other variants (P < 0.05). Measured values of the availability of mineral nitrogen and plant biomass production indicate the importance of species composition at the establishment of new grassland. For example, species composition of permanent grassland has a great importance at the establishment of new grassland on erosion-affected soils in protection areas of drinking water. Because selected species of grasses can immobilize nitrogen in their bodies, thus preventing it’s leaching from the soil.