Inoculation with beneficial microbes represents a promising solution for sustainable agricultural production; however, knowledge on the effects of inoculants on the indigenous microbial communities remains limited. Here, we evaluated the impact of the arbuscular mycorrhizal fungus Rhizophagus irregularis and the promoting rhizobacterium Bacillus subtilis on the growth of Lactuca sativa. The biomass, the composition, and the enzyme activity (urease, acid phosphatase, and β-glycosidase) of the rhizosphere microbial community at two soil moisture levels (5 and 10% soil water content) were evaluated. Fungal colonization was lower in co-inoculated plants than those only inoculated with R. irregularis. Plant growth was enhanced in co-inoculated and B. subtilis inoculated soils. Bacterial biomass and the composition of the microbial communities responded to the joint effect of inoculant type × water regime while the biomass of the other microbial groups (fungi, actinomycetes, microeukaryotes) was only affected by inoculant type. Co-inoculation enhanced the activity of acid phosphatase, indicating a synergistic effect of the two inoculants. Co-inoculation positively impacted the index reflecting plant–microbial soil functions under both water regimes. We concluded that the interactions between the two inocula as well as between them and the resident rhizosphere microbial community were mainly negative. However, the negative interactions between R. irregularis and B. subtilis were not reflected in plant biomass. The knowledge of the plant and rhizosphere microbial responses to single and co-inoculation and their dependency on abiotic conditions is valuable for the construction of synthetic microbial communities that could be used as efficient inocula.