Sour cherry (Prunus cerasus L.) is one of the most important fruit crops in Poland and ‘Łutówka’ is the dominant cultivar in commercial orchards. The experiment was carried out in 2007–2013 in three orchards; in each of them, three levels of fertilization were applied: 0 N kg ha−1, 60 kg N ha−1, and 120 kg N ha−1. The activity of dehydrogenase and protease in the soil was studied depending on nitrogen fertilization. The abundance of soil microorganisms was assessed: bacteria, fungi, actinomycetes, and nitrogenous bacteria (Azospirillum and Azotobacter) in the years during the experiments carried out with fertilization of 60 kg N ha−1 in all orchards. The enzyme activity of dehydrogenases increased after the use of 60 kg N ha−1 from 3.8 to 6.7 (cm3 H2 24 h−1 kg−1 DW soil), but a further increase in the dose to 120 kg N ha−1 caused a decrease in activity to 5.1 (cm3 H2 24 h−1 kg−1 DW soil). The activity of proteases was dependent on nitrogen fertilization, but to a large extent it was related to the course of climatic conditions. There is no relationship between the growth and the activity of proteases. The yield and selected quality parameters of the cherry fruits were associated with both dehydrogenases and proteases. The use of lower doses of nitrogen fertilizers allows for maintaining biological balance in the soil and a more efficient use of nutrients, contributing to less environmental pollution.