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The natural soil N supply in volcanic soils (Andisols) can be a significant source of plant-available N for agro-ecosystems. Nevertheless, intensive farming systems in south Chile apply high fertilization rates, which lead to high production costs and involve a risk for adverse ecosystem effects. In order to achieve sustainable land management, a better understanding of the processes that govern soil N availability and loss, and their external drivers, is required. In this study, we selected a winter-cropland, a summer crop-winter fallow rotation, and a forest, used as a reference ecosystem. Gross N transformations (15N isotope dilution) and microbial community structure (phospho-lipid fatty acid analysis) in the topsoil were determined. Gross N mineralization was about ten times lower in the agro-ecosystems than in the forest, while gross nitrification was low in all sites. Gross N immobilization equalized or exceeded the gross inorganic N production in all sites. Microbial biomass was 3–5 times more abundant in the forest than in the agro-ecosystems. A positive relationship between the ratio fungi/bacteria and total microbial biomass was observed in these Andisols. We suggest that the reduction in fungal biomass induced a lower extracellular enzyme production and limited soil organic matter depolymerisation in the agro-ecosystems. We conclude that soil N cycling was unable to provide a significant N input for the croplands, but also the risk for ecosystem N losses was low, even under fallow soil conditions. Current fertilization practices appropriately anticipated the soil N cycling processes, but further research should indicate the potential of alternative land management to reduce fertilizer cost.