The ongoing scientific debate on the selection of the best bioindicators to reflect the quality of arable soils indicates both their microbiome and biochemical parameters. Consideration has also been given to the fact that Zea mays has achieved the status of a crop used in the feed industry and for energy purposes, and Triticosecale is attracting increasing interest in this area. Therefore, the aim of this study was to determine the wide range of effects of Zea mays and Triticosecale cultivation on soil microbial and biochemical activity. The assessment of these parameters was based on the determination of microbial abundance, colony development index (CD), ecophysiological index of microbial diversity (EP), soil enzyme activities (dehydrogenases, catalase, urease, acid phosphatase, alkaline phosphatase, β-glucosidase, and arylsulfatase) as well as soil physicochemical properties. The innovative nature of the research was achieved by extending the pool of analyses to include both microbial biodiversity and analysis of soil samples at three depths: 0–20 cm; 21–40 cm; and 41–60 cm. It was found that the highest activities of soil enzymes and the abundance of organotrophic bacteria and fungi, as well as their colony development indices (CD), occurred within the rhizosphere and that their values decreased with increasing depth of the soil profile layers. Two phyla, Actinobacteria and Proteobacteria, representing the microbiome of arable soils, were identified independently of soil management practices. Unique bacterial genera in the soil under Triticosecale cultivation were Pseudonocardia, whereas Rhodoplanes, Nocardioides, and Rhodanobacter were found under Zea mays cultivation. The activity of all enzymes, especially urease and arylsulfatase, was significantly higher in the soil under Triticosecale. This was influenced by the more favorable physicochemical properties of the soil.