The profound intensification of agricultural practices by increased application of agro-chemicals, short crop rotations and ploughing resulted in loss of soil fertility, erosion and accumulation of soil-borne plant pathogens. Soil microbial communities are key players in ecosystem processes and are intimately linked to crop productivity and health. Thus a better understanding of how farming practices affect soil microbiota is needed in order to promote sustainable agriculture. The long-term field trial in Bernburg (Germany) established in 1992 provides a unique opportunity to assess the effects of i) the crop (maize vs. rapeseed) preceding the actual winter wheat culture, ii) tillage practice (mouldboard plough vs. cultivator tillage) and iii) standard nitrogen (N)-fertilization intensity with application of growth regulators and fungicides (intensive) compared to reduced N-fertilization without growth regulators and fungicides (extensive). We hypothesized that these different farming practices affect the soil prokaryotic community structures with consequences for their functional potential. Total community-DNA was extracted directly from soils sampled at wheat harvest. Illumina sequencing of 16S rRNA genes amplified from total community-DNA revealed a significant effect of tillage practice and the preceding crop on prokaryotic community structures, whereas the influence of N-fertilization intensity was marginal. A number of differentially abundant prokaryotic genera and their predicted functions between mouldboard plough vs. cultivator tillage as well as between different preceding crops were identified. Compared to extensive N-fertilization, intensive N-fertilization resulted in higher abundances of bacterial but not of archaeal amoA genes, that are involved in ammonia oxidation. Our data suggest that long-term farming strategies differently shape the soil prokaryotic community structure and functions, which should be considered when evaluating agricultural management strategies regarding their sustainability, soil health and crop performance.