Understanding plant microbiomes may offer new insights into plant disease management. Fusarium head blight (FHB), caused by Fusarium graminearum, is a serious disease of wheat and barley that impacts both grain yield and quality via mycotoxin contamination. We hypothesized that differential enrichment in the barley spike in response to disease could identify plant-recruited microbiota with the potential to inhibit FHB. Selective breeding of barley lines may then be used to harness the barley spike microbiome in a beneficial manner. We analyzed the composition of the barley spike microbiome during FHB disease onset from ten genotypes of a breeding population grown in four FHB nurseries (ID, MN, ND, and NY). Bacteria and fungi were identified using amplicon sequencing and analyzed for their responsiveness to disease and genotype. Barley genotype significantly affected bacterial community composition at each location, while disease had a significant effect only in MN, where disease pressure was highest. There was also a significant genotype by disease effect at three locations. The microbiome at each location contained plant-beneficial taxa, including Alternaria, Cryptococcus, Pantoea, Pseudomonas, Pseudozyma, and Sphingomonas, among others. High-throughput bacterial culturing on 2022 samples resulted in the collection of ~2,000 isolates. These will be used to generate synthetic communities to test microbiome drivers by host genotype and the ability to protect against FHB.