In the present study, biological indicators were used to assess the impact of applying spent mushroom substrate and manure on the soil environment. The use of spent mushroom substrate had a varied effect on the microorganisms. Stimulation was recorded in the abundance of copiotrophic bacteria and fungi, but only in the first year of the study. In the case of cellulolytic bacteria, this effect was visible only in single plots. Similar observations were also noted regarding the relative DNA content (in relation to the control), which increased for both bacteria and fungi after applying spent mushroom substrate. In the soil fertilized with spent mushroom substrate, a decrease in DNA concentration was observed, but only in the first and second year. For enzymatic activity, the use of spent mushroom substrate alone proved to be more favorable, but this effect was again observed only in the first year of the study. The application of manure caused similar changes as observed with the use of spent mushroom substrate. These observations indicate a similar impact of spent mushroom substrate and manure on the parameters tested. The research presented suggests the use of both classical methods and methods based on the analysis of DNA extracted from soil to study the impact of spent mushroom substrate on the activity of soil microbial populations.