Mineral and organic fertilizers are the important factors for maintenance and improvement of soil fertility and aggregation. Despite aggregation and aggregate stability are proxy of soil fertility, the connection between fertilization and aggregation is not direct, as short and long-term processes may affect the aggregate formation in different directions. In this study, the long-term effects of a 20-year application of mineral and organic fertilizers were studied in an intensive horticultural crop rotation with the following treatments: i) without fertilization (control soil), ii) nitrogen applied by mineral fertilizer, and iii) farmyard manure application with low (30 t ha −1 y −1) or iv) high (60 t ha −1 y −1) rates. In case of short-term aggregation process, K-polyacrylate was added to the soil to change aggregate composition and then the aggregated soils were incubated for 2 weeks. Long-term fertilization increased the soil organic C (SOC) content by 42–73% and the portion of small macroaggregates (1–0.25 mm) compared to control soil. In contrast, soil aggregation induced by K-polyacrylate showed an increase of the large macroaggregate (2–1 mm) portion independent of fertilization. Polyacrylate had no effect on soil microbial biomass C. According to the increased SOC content, the fertilization increased CO 2 efflux from soil (4.2–5.2% of SOC after 80 days of incubation). Short-term aggregation by K-polyacrylate decreased the SOC mineralization rate mainly of the labile C-pools. In conclusion the data of this study suggest that long-term fertilization mainly contributes to the formation of small macroaggregates. In contrast, the formation of large macroaggregates is mediated mainly by short-term processes and contributes to the decrease of SOC mineralization.