Sustainable soil management of fruit orchards can have positive effects on both soils and crop yields due to increases in microbial biomass, activity and complexity. The aim of the present study was to investigate medium-term effects (12 yr) of two different management practices termed ‘sustainable’ (ST) and ‘conventional’ (CT) on soil microbial composition and metabolic diversity of a rainfed mature olive orchard in Southern Italy. ST included no-till, spontaneous self-seeding weeds (mainly graminaceous and leguminosae)spontaneous graminaceos and cover crops, and mulch derived from olive tree prunings, whilst CT was managed by frequent tillage and included severe pruning with residues removed from the orchard. Microbial analyses were carried out by culture-dependent methods (microbial cultures and Biolog®). Molecular methods were used to confirm the identification by light microscopy of the isolates of fungi and Streptomyces. A significant higher number of total culturable fungi and bacteria was found in ST. The number of fungal groups in ST was also significantly greater than in CT. Overall and substrate-specific Biolog® metabolic diversity indices of microbial communities and soil enzyme activities were greater in ST. The results demonstrate that soil microorganisms respond significantly to sustainable orchard management characterized by periodic applications of locally derived organic matter. This study confirms the need for Mediterranean orchards to encourage farmers to practice soil management based on organic matter inputs associated with zero tillage in order to improve soil functionality.