Five soils characterised by different agro-forest managements, typical of Mediterranean environment and with increasing human impact were chosen in Sardinia (Italy): two vineyards with different management systems, a rotation hay crop-pasture and a forest (Quercus suber L.). The study aimed to investigate the relationships between C storage and microbial functionality in soil under different managements. Pools of total organic C and microbial biomass C were determined, as well as the loss of organic C due to microbial respiration (basal and cumulative) and several microbial indices (metabolic, mineralization, and microbial quotient) as indicators of the microbial efficiency in the use of energy and the degree of substrate limitation for soil microbes. Enzymes were chosen on their relevance in the C (β-cellobiohydrolase, N-acetyl-β-glucosaminidase, β-glucosidase, α-glucosidase), N (leucine aminopeptidase), S (arylsulphatase) and P (acid phosphatase) cycling and were used as indicators of functional diversity in soil. Organic C pools and enzyme activities on average increased noticeably in soils with a lower human impact showing the highest values in forest and the lowest in the vineyards, following the trend of organic matter availability. The trend in functional diversity reflected the increase of microbial pool and organic C availability: the vineyards showed a lower Shannon’s diversity index, whilst pasture and forest sites reached the maximum levels of functional diversity. These soils showed an increase of microbial efficiency in the use of available resources and the decrease of substrate limitation for soil microbes.