This ready to load phyloseq R S4 object contains the OTU table, taxonomy table and sample metadata. This data was build using kraken v2.1.2, kraken-biom v1.0.1 and phyloseq version 1.4. Sequencing data is deposited at NCBI-SRA under BioProject: PRJNA952340. Study abstract Paul B. Stege, Dirkjan Schokker, Frank Harders, Soumya K. Kar, Norbert Stockhofe, Vera Perricone, Johanna M. J. Rebel, Ingrid C. de Jong, Alex Bossers Modern broiler breeds allow for high feed efficiency and rapid growth, which come at a cost of increased susceptibility to pathogens and disease. Broiler growth rate, feed efficiency, and health are affected by the composition of the gut microbiota, which in turn is influenced by diet. In this study, we therefore assessed how diet composition can affect the broiler jejunal gut microbiota. A total of 96 broiler chickens were divided into four diet groups: control, coated butyrate supplementation, medium-chain fatty acid supplementation, or a high-fibre low-protein content. Diet groups were sub-divided into age groups (4, 12 and 33 days of age) resulting in groups of 8 broilers per diet per age. The jejunum content was used for metagenomic shotgun sequencing to determine the microbiota taxonomic composition at species level. The composed diets resulted in a total of 104 differentially abundant bacterial species. Most notably were the butyrate-induced changes in the jejunal microbiota of broilers 4 days post-hatch, resulting in the reduced relative abundance of mainly Enterococcus faecium and the opportunistic pathogen Enterococcus hirae, when compared to the control diet. This effect takes place during early broiler development, which is critical for broiler health, thus exemplifying the importance of how diet can influence the microbiota composition and its relation to broiler health. Future studies should therefore elucidate how diet can be used to promote a beneficial microbiota in the early stages of broiler development.