Beneficial bacteria in the rhizosphere are known to trigger faster and stronger plant immune responses to biotic and abiotic stressors. In the present study, we aimed to test the hypothesis that a rhizosphere microbiome transplant (RMT) may improve the immune response and reduce the disease rates of barley ( Hordeum vulgare). This hypothesis was tested in a greenhouse system with the powdery mildew-causing fungus Blumeria graminis f. sp. hordei ( Bgh). Detached rhizosphere microbiome from barley grown in a field soil was transplanted to barley seedlings grown in potting soil with reduced microbial diversity. Saline-treated plants served as control. At the three-leaf stage, barley was infected with Bgh. Decreased susceptibility to Bgh was observed for barley treated with the RMT as displayed by lower Bgh pustule counts in a detached leaf assay. A trend toward enhanced relative transcript abundances of the defense-related genes PR1b and PR17b was observed in leaves, 24 h after the Bgh challenge, when compared to the control. Moreover, 10 days after the Bgh challenge, the barley rhizosphere microbiome was harvested and analyzed by sequencing of 16S rRNA gene amplicons. The microbial community composition was significantly influenced by the RMT and displayed higher microbial diversity compared to the control. Furthermore, microbial beta-diversity and predicted functional profiles revealed a treatment-dependent clustering. Bacterial isolates from the RMT showed in vitro plant beneficial traits related to induced resistance. Our results showed that transplantation of a rhizosphere microbiome could be a sustainable strategy to improve the health of plants grown in potting soil with low microbial diversity under greenhouse conditions.