Microbially mediated soil organic matter is an extremely sensitive pool that indicates subtle changes in the quality parameters responsible for the soil’s ecological and productive functions. Fifty years of mineral fertilization of a wheat-corn cropping system has a strong impact on soil quality parameters. The goal of the research was to study the dynamics and quality of soil biological parameters affected by increasing amounts of mineral nitrogen. Soil respiration, potentially mineralizable C and N, microbial biomass C and N and light-fraction OM on Cambisol were analyzed in the following treatments: (1) Control (without fertilization); (2) NPK (60/51/67); (3) NPK (90/51/67); (4) NPK (120/51/67); (5) NPK (150/51/67 kg ha−1). The parameters studied were significantly affected by the long-term application of mineral fertilizer compared with both the control and the adjacent native soil. The highest amounts of nitrogen (N150) did not significantly differ from N120 and N90 for most of the parameters studied. Potentially mineralizable C represented the largest labile carbon pool, while microbial biomass N was the largest labile nitrogen pool. The mineralization rates for C and N were oppositely distributed over the seasons. The sensitivity index correlated with the amount of light-fraction OM. The results give a deeper insight into the behavior and distribution of different pools of labile SOM in the agro-landscapes and can serve as a reliable basis for further research focused on zero soil degradation.