The implementation of cover crops into a crop rotation can contribute to a more sustainable soil management. For the improved decomposition of cover crop residues, the commercial inoculant Effective Microorganisms® (EM) is increasingly applied. Despite its extensive application, comprehensive studies on the effect of EM application on soil processes are lacking, since rarely a clean differentiation between an EM-effect (induced by living EM directly) or a substrate effect (induced by the accompanying EM substrate) is made. To determine the potential effects of EM application after cover crop integration to soil we conducted a lab incubation experiment under spring-like conditions in temperate climates and applied EM either on bare soil or on cover crops prior to soil incorporation at recommended and 100 times the recommended doses. Control groups included treatments with no EM addition and a sterilized EM solution applied at 100 times the recommended dose. Over a monitoring period of 28 days, the application of EM at the recommended dose showed no consistent effect on soil respiration, microbial bound carbon or nitrogen, soil pH, permanganate oxidizable carbon or water extractable nutrients and trace elements. Any observed effects in the treatment that received 100 times the recommended dose was attributed to the substrate introduced with the EM solution rather than the living EM themselves. Amplicon sequencing showed that certain EM taxa could be detected in soil at low abundance after EM application, but only when EM were applied at 100 times the recommended dose. We conclude that the application of EM did not produce a discernible effect on soil biological or chemical properties, nor did it influence the decomposition process of the cover crop.