The long-term effects of biogas slurry application on soil fertility indices were compared with raw slurry in biodynamic organic farming systems. An on-farm soil and slurry sampling was carried out to quantify the effects on stocks of soil organic matter, microbial biomass and microbial residues. Five fields with biogas slurry and five neighbouring fields with raw slurry amendments were selected at 5 different sites in the north-east of Baden-Württemberg, Germany. The application of biogas slurry ranged from 15 to 25 years and did not affect SOC, total N stocks or the soil C/N ratio. Biogas slurry application decreased the soil microbial biomass to SOC ratio, which indicates a reduced availability of the biogas slurry C input to soil microorganisms compared with raw slurry. At some sites, differences in clay content masked any slurry effects on the microbial activity, biomass, and residue indices. There were no general effects of biogas slurry on the ratios of ergosterol to microbial biomass C or amino sugar-based fungal C to bacterial C, whereas an increasing clay content caused a significant shift towards bacteria according to the latter ratio. Since the soils had been farmed organically in diverse crop rotations for at least 40 years, chemical differences in slurry composition were not great enough to result in different biochemical properties. The consistency in the data of all approaches strongly indicates the validity of the current on-farm study by comparing neighbouring fields.