The constant increase in the intensity of agricultural production simultaneously increases the risk of negative effects of long-term agricultural practices. By-products of agricultural, forestry, and food production, as well as other types of organic waste, can be used as raw materials in the production of organic fertilizers and substrates for seedling cultivation through various processes of biological stabilization. In this way, the amount of waste is reduced, which contributes to the preservation of soil fertility and the sustainable use of resources. During waste processing and the stabilization of organic matter can be improved by using earthworms (vermicomposting). The aim of this study was to determine how different substrates, composed of different components and their mixtures, affect the earthworm Eisenia andrei. The effects of investigated substrates on the survival and behavior of earthworms were monitored. In addition, the effect of tested substrates on acetylcholinesterase (AChE), carboxylesterase (CES), and glutathione S-transferase (GST) activity was also assessed. The results showed that the most suitable substrates were leaves with horse manure and grape pomace alone and in combination with rock wool and sawdust. The obtained results provide important information on components and mixtures that have the greatest potential in the production of organic fertilizers and substrates for growing seedlings.