The intensification of agriculture leads to worrying changes in agro-ecosystems. Research has been conducted to bridge the gap between the desire to maintain ecological balance and harmful interference with ecosystems. Spent mushroom substrate (SMS) can become the basis of a farming system that improves soil quality. The aim of the study was to assess the potential of SMS in improving the following soil quality indicators: abundance and activity of microorganisms, and to assess the impact of SMS and manure (M) on the increase in the greenhouse effect. The plots were fertilized with SMS, M, and SMS in combination with NPK mineral fertilization. The application of SMS had a varied but generally positive effect on the parameters studied, particularly on the number of proteolytic microorganisms, urease activity but also ammonification and nitrification. In contrast, inhibition of protease activity was observed. The stimulation of most of the indicators was recorded in the first and second years, followed by a weakening of their effect. M also positively influenced the tested parameters, especially nitrification, where this effect lasted longer than for SMS. This indicates that the application of manure contributes more to the formation of products from which denitrification can potentially generate greenhouse gases