Intensive agriculture based on repeatedly plowing a monoculture is known to degrade the structural, chemical, and biological properties of soils. Conservation agriculture is gaining popularity worldwide with practices including reduced tillage, increased plant diversity, and implementation cover crops. The aim of this study was to assess the impact of crop rotation and cover crops on soil microbial communities. The hypothesis was that enhanced plant diversity would boost the diversity of the soil microbiome. Two rotation were tested by adding a cereal (corn [Zea mays L.]soybean [Glycine max (L.) Merr.] and corn-soybean-wheat [Triticum aestivum L.]), as well as the implementation of cover crops. The enhanced plant diversity had no impact on total molecular biomass. The bacteria/fungi ratio varied across the plots but was not clearly linked to the enhanced plant diversity. Bacterial richness was not influenced by the treatments, whereas eukaryotic richness decreased in the presence of cover crops in one of the sites. Microbial composition was the most sensitive indicator to enhanced plant diversity. Differential relative abundance (log2 fold changes) identified proteobacterial amplicon sequence variants (ASVs) specifically related to each crop rotation system and to the presence of cover crops. There was more ASV of Actinobacteria associated with the three-crop rotation system and less ASV of Acidobacteria associated with the cover crops system compared to the two-and three-crop rotation systems, respectively. As for eukaryotes, the number of ASVs belonging to Ascomycota and Cercozoa phyla and associated with the three-crop rotation system is less important than for the two-crop rotation system. This study shows that conservation agricultural practices can influence soil microbial communities. The variations in some ASVs could have functional implications on organic matter decomposition or plant growth and in terms of soil ecosystem services and field crop sustainability.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.