Organic amendment and tillage reduction are two common practices to contrast soil organic matter decline, thus promoting sustainable cropping and carbon sequestration. In a horticultural land use system under Mediterranean climate, we evaluated the 9-year effects of two compost inputs (15 and 30 t ha−1 y−1, low and high input, respectively) and two tillage intensities (intensive and reduced) on soil macronutrients concentration, microbial biomass and activity. Total organic C, total N and POlsen were smaller in plots amended at low input, whilst intensive tillage decreased them at both compost inputs. These decreases in intensively tilled plots was ascribed to the disruption of soil aggregates, with consequent microbial degradation of the physically protected organic matter by oxidative processes. On the contrary, reduced tillage increased the extractable C, likely due to a higher protection of the most labile soil C fraction from the mineralization. Similarly, microbial biomass C and N increased following both doubling compost input and reducing tillage intensity, with a greater effect by the first factor. The higher values of cumulative 10-day basal and 20-h glucose-induced respiration, and metabolic quotient in intensively tilled plots suggested that high tillage favoured soil aggregate disruption and C accessibility. This was also confirmed by higher values of dehydrogenase activity/total organic C in those plots. Intensive tillage caused a higher soil aeration and organic substrates accessibility to microflora, thus undoing the fertility benefits provided by the high compost input. However, also a low compost input coupled to reduced tillage seemed to accomplish soil sustainability needs.