The dynamic microbiota in chickens can be affected by exposure to antibiotics, which may alter the composition and substrate availability of functional pathways. Here, 120 Jing Hong chicks at 30 days of age were randomly divided into four treatments totaling seven experimental groups: control chicks not exposed to antibiotics; and chicks exposed to enrofloxacin, diclazuril, and their mixture at 1: 1 for 14 days and then not exposed for a withdrawal period of 15 days. Fecal samples were collected from the 7 groups at 8 time-points (exposure to 4 antibiotics and 4 withdrawal periods) to perform in-depth 16S rRNA sequencing of the gut microbiota. Taxon-independent analysis showed that the groups had significantly distinct microbial compositions (p< 0.01). Based on the microbial composition, as compared with the control group, the abundances of the phyla Firmicutes, Actinobacteria, Thermi, and Verrucomicrobia, as well as the families Lactobacillus, Lactococcus, S24-7, and Corynebacterium, were decreased in the antibiotic-exposed chicks (p< 0.01). Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analyses revealed significant differences in microbiota metabolite pathways due to the genera of the antibiotic-responsive microbes (p< 0.01), especially the pathways relating to cell growth and death, immune system diseases, carbohydrate metabolism, and nucleotide metabolism. Oral treatment with enrofloxacin, diclazuril, and their mixture modified the gut microbiota composition and the microbial metabolic profiles in chickens, with persistent effects (during the withdrawal period) that prevented the …