Microbial processes mediated by soil enzymes are crucial in soil organic matter decomposition, resulting in release of nutrients that become available for plant and microbial uptake. Therefore, it is crucial to know the sensitivity of enzyme activities (EA) along soil depths at distinct plant vegetation stages, and how the availability of mineral nitrogen (N) alters EA. We studied effects of N fertilization (0 and 160 kg N ha−1), soil depth (0–35 cm), and plant-phenological stage (silking and maturity) on microbial biomass C (Cmic) and potential activities of C-, N- and P-acquiring enzymes in the field under Zea mays L. Nitrogen fertilization increased shoot biomass by more than 80% compared to unfertilized plants. Maize roots triggered increases in Cmic and EA for all measured enzymes compared to bare fallow. Stimulating effect of plant roots on EA was enzyme specific and stronger at silking than maturity stage of maize. The down-regulating effect of N fertilization on EA involved in acquiring N was most pronounced on the activity of L-leucine aminopeptidase and β-1,4-N-acetylglucosaminidase. Soil depth was the primary determinant of EA, explaining up to 51% of the variation. Depth-dependent EA changes were stronger in rooted soil. A pronounced biotic control on EA was demonstrated by higher EA in rooted soil than in bare fallow. This confirmed root-mediated microbial activation. Stronger effect of silking vs. maturity stage on EA indicated that actively growing roots fuel microorganisms via root-derived organics. Thus, soil depth and plant roots were major factors controlling microbial activity in arable soil.