Diversification of agricultural practices, including changes in crop rotation, intercropping or cover cropping, influence the soil microbiome. Here the impact of tillage and crop diversification on the soil microbiome is reported, being one of the few boreal studies. The field experiment consisted of four treatments with four replications all having a short cereal rotation practice namely an oat (Avena sativa) – spring barley (Hordeum vulgare) – wheat (Triticum aestivum) rotation for the past 10 years until spring 2018. During that period two of the treatments were conventionally tilled with moldboard ploughing whereas the other two were no-tillage treatments. From the growing season 2018 until fall 2020 the main crop in all treatments was spring barley. The first conventional tillage treatment was diversified with English ryegrass (Lolium perenne) as an undersown cover crop for the next three growing seasons. The first no-tillage treatment continued with spring barley only. The second conventional tillage and no-tillage treatment had winter rapeseed in rotation in 2019. Bulk soils were sampled in May 2018 before diversification and then in October 2018, 2019, and 2020. The results showed a clear effect of tillage on the beta-diversity of the soil microbiome and an increase in fungal richness. Barley monoculture interrupted with winter rapeseed resulted in a minor change of the fungal and bacterial community composition. Other fungal and bacterial alpha diversity measures did not react to tillage or diversification nor did the gene copy abundances involved in the N cycle. In conclusion tillage had a profound effect on the soil microbiome hindering impact of the diversification.