Winter wheat is an important cereal consumed worldwide. However, current management practices involving chemical fertilizers, irrigation, and intensive tillage may have negative impacts on the environment. Conservation agriculture is often presented as a sustainable alternative to maintain wheat production, favoring the beneficial microbiome. Here, we evaluated the impact of different water regimes (rainfed and irrigated), fertilization levels (half and full fertilization), and tillage practices (occasional tillage and no-tillage) on wheat performance, microbial activity, and rhizosphere- and root-associated microbial communities of four winter wheat genotypes (Antequera, Allez-y, Apache, and Cellule) grown in a field experiment. Wheat performance (i.e., yield, plant nitrogen concentrations, and total nitrogen uptake) was mainly affected by irrigation, fertilization, and genotype, whereas microbial activity (i.e., protease and alkaline phosphatase activities) was affected by irrigation. Amplicon sequencing data revealed that habitat (rhizosphere vs. root) was the main factor shaping microbial communities and confirmed that the selection of endophytic microbial communities takes place thanks to specific plant–microbiome interactions. Among the experimental factors applied, the interaction of irrigation and tillage influenced rhizosphere- and root-associated microbiomes. The findings presented in this work make it possible to link agricultural practices to microbial communities, paving the way for better monitoring of these microorganisms in the context of agroecosystem sustainability.