The understanding of agro-ecosystems is the key to estimating the influence of long-term agricultural production methods on the environment. The present study aimed to improve the knowledge of functional and structural soil microbial diversity influenced by differentiated fertilization at four German long-term field experiments. Soil microbial biomass, β-glucosidase, protease, alkaline phosphatase, DMSO-reduction and TTC-dehydrogenase were evaluated in soils from three different fertilizer treatments (control, farmyard manure, mineral fertilizer) at four sites (Lauterbach, Bad Lauchstädt, Halle, Thyrow). Profiles of 29 identified phospholipid fatty acids were used for the evaluation of structural diversity. The results obtained showed that fertilization applied over several decades or even more than a century has led to distinct alterations of the soil ecosystem. There was a clear separation of the sites by microbial biomass and metabolic activities into dependence of soil properties and fertilization treatments. In contrast to the control, microbial biomass and enzyme activities of the FYM soils increased more strongly than in the NPK plots. The results of PLFA analyses indicate that the community of the autochthonous microflora has differently changed among sites and fertilizer application. Principle component analyses revealed that bacteria especially Gram-positive bacteria and eukaryotes were responsible for these differences.