No-tillage (NT) and reduced tillage (RT) systems are well-known management tools for reducing soil erosion and improving soil fertility. NT and RT may improve the environmental and economic performance of organic farming, but they are still not common practice among organic farmers. This paper presents the effects of tillage [RT versus conventional tillage (CT)], fertilization (slurry versus manure compost) and biodynamic preparations (with versus without) on soil fertility indicators such as soil organic carbon (Corg), microbial biomass and microbial activity, soil nutrients and nutrient budgets in an organic farming system during the first six-year crop rotation period of a long-term experiment on a clayey soil in a temperate climate. RT caused stratification of soil organic carbon (Corg), microbial properties and soil nutrients in the soil profile. Under RT, Corg in the 0–10 cm soil layer increased from 2.19 to 2.61% (w/w) from 2002 to 2008, whereas it remained constant under CT. In both tillage treatments, Corg remained constant in the 10–20 cm soil depth. Microbial biomass C increased by 37% under RT in the 0–10 cm soil depth and microbial activity [dehydrogenase activity (DHA)] was enhanced by 57%. Soil microbial biomass C and DHA in the 10–20 cm soil depth were also higher under RT (+10 and +17%, respectively). Soluble soil P and K were 72 and 40%, respectively, higher in 0–10 cm soil depth under RT when compared with CT. Fertilization showed no effects on the measured soil properties. Biodynamic preparations increased solely the Cmic-to-Nmic (soil microbial biomass C to soil microbial biomass N) ratio by 7% in the 0–10 cm soil depth. Nutrient budgets for P were balanced in all treatments, but N and K exports were higher under RT compared to CT. We conclude that RT is a suitable method for increasing indicators of soil fertility in organic farming systems. The combined effects of RT and an organic farming system with a diverse, ley-based crop rotation and organic fertilization merit further promotion and it may be considered for supporting actions by the agricultural policy schemes.