ABSTRACT Long-term agricultural practices are assumed to shape the rhizosphere microbiome of crops with implications for plant health. In a long-term field experiment, we investigated the effect of different tillage and fertilization practices on soil and barley rhizosphere microbial communities by means of amplicon sequencing of 16S rRNA gene fragments from total community DNA. Differences in the microbial community composition depending on the tillage practice, but not the fertilization intensity were revealed. To examine whether these soil and rhizosphere microbiome differences influence the plant defense response, barley (cultivar Golden Promise) was grown in field or standard potting soil under greenhouse conditions and challenged with Blumeria graminis f. sp. hordei (Bgh). Amplicon sequence analysis showed that preceding tillage practice, but also aboveground Bgh challenge significantly influenced the microbial community composition. Expression of plant defense-related genes PR1b and PR17b was higher in challenged compared to unchallenged plants. The Bgh infection rates were strikingly lower for barley grown in field soil compared to potting soil. Although previous agricultural management shaped the rhizosphere microbiome, no differences in plant health were observed. We propose therefore that the management-independent higher microbial diversity of field soils compared to potting soils contributed to the low infection rates of barley.