An understanding of agroecosystems is the key to estimate the influence of agricultural production methods on the environment, including soil, water and air quality. The present study aimed to contribute to the insufficient knowledge of functional and structural soil microbial properties influenced by organic and inorganic fertilisation and climatic conditions at two European long-term field experiments. Soil microbial biomass, activities of alkaline phosphatases, β-glucosidases and proteases, and phospholipid fatty acid profiles were evaluated in soils from three different fertiliser treatments (inorganic, NPK; farmyard manure, FYM; without, CON) at two sites (Bad Lauchstädt, Germany and Keszthely, Hungary). The results showed that C_org and N_t concentrations were significantly higher in the FYM plots compared to the NPK amendments or CON. Furthermore, increases were observed in the C_mic and the enzyme activities of FYM-fertilised treatments in the soil at Bad Lauchstädt. NPK fertilisation significantly decreased C_mic in the soil at Keszthely, whereas FYM increased the activity of β-glucosidases. Ecophysiological parameters (C_mic-to-C_org and qCO₂) revealed a clear climatic influence on edaphic properties. The profiles of 28 phospholipid fatty acids (PLFAs) were used to assess whether observed changes in functional microbial parameters were accompanied by changes in the composition of the microbial communities after FYM and NPK amendments. The results of principal component analyses indicate that the communities of the autochthonous microflora differed between the two sites and three treatments. Bacteria, particularly Gram-negative bacteria and eukaryotes were responsible for these differences. The results support the hypothesis that fertilisation in general and its forms (organic versus inorganic) affect important functional and structural soil microbial properties. PLFA analysis proved to be a more sensitive indicator than functional parameters.