Intensive agronomic practices such as deep and repeated tillage and applying high mineral fertilization rates to improve crop yields have gradually determined soil resource degradation. A study was carried out over a two-year period (2015 and 2016) to assess effects of tillage (plough; subsoil; and spading) and fertilization (mineral vs. organic) on soil health relative to carbon and nitrogen dynamics in potato crop in the Mediterranean environment. Microbial indices could be successfully used as tool for assessing soil health in terms of predictors and indicators of carbon sequestration and nitrogen availability. The microbial quotients, calculated as percentage of the microbial-C to total organic C (C_mic:C_org), was significantly higher in subsoiling than in plowing and spading soil tillage, and higher in 2016 (3.19%) than 2015 (1.72%). The activity of enzymes involved in C cycle was significantly higher in subsoiling and spading than in plowing, while acid phosphatase was positively affected by spading and arylsulfatase increased with plowing. The whole enzyme activity expressed as synthetic enzymatic index (SEI) was positively affected by subsoiling and plowing in 2015 (4254) compared to spading tillage (3934). A general decrease in soil enzyme activity in 2016 than 2015 was observed. The subsoiling in potato crop favored the immobilization of carbon and nitrogen during the wet spring–summer period. Conversely, the plowing favored the mineralization process when the spring–summer period became more dried.