Flooding is a predominant abiotic stress for cultivated plants, including barley. This cereal crop shows a large adaptability to different environmental conditions, suggesting the presence of key traits to tolerate adverse conditions. During germination, genetic variations account for dissimilarities in flooding tolerance. However, differences in the seed microbiota may also contribute to tolerance/sensitivity during seedling establishment. This work investigated differences in microbiome among the grains of barley accessions. Two barley phenotypes were compared, each either tolerant or sensitive to a short submergence period followed by a recovery. The study used a metataxonomic analysis based on 16S ribosomal RNA gene sequencing and subsequent functional prediction. Our results support the hypothesis that bacterial microbiota inhabiting the barley seeds are different between sensitive and tolerant barley accessions, which harbour specific bacterial phyla and families. Finally, bacteria detected in tolerant barley accessions show a peculiar functional enrichment that suggests a possible connection with successful germination and seedling establishment.