Although concerns about the impacts of antibiotics in aquatic organisms are reported worldwide, the potential adverse effects on fish gut microbial communities and fish health are still not well known. In this study, we investigated the effects of florfenicol (FFC) on the gut microbiome and gastrointestinal (GIT) gene expression in juvenile Atlantic salmon (Salmo salar). Three doses of FFC were used to coat experimental feed at 10, 20 and 30 mg/kg/ fish body weight (bw). The feed was administered for 18 days, followed by a 10-day recovery period. The metatranscriptome analysis revealed that 10 and 30 mg/kg bw of FFC led to the downregulation of genes involved in the transcription of NADH-ubiquinone oxidoreductase chain-1, suggesting that the antibiotic targets bacterial respiratory metabolism. The 30 mg/kg bw FFC treatment upregulated genes that encode glycolytic enzymes, such as phosphoglycerate kinase, indicating a disruption of energy metabolism in the microbiome. Analysis of the fish host transcriptome showed that the FFC treatment affected cellular processes in the GIT system of fish, including pathways related to apoptosis and DNA metabolism. The 30 mg/kg bw FFC treatment specifically activated pathways related to cellular regulation, including LXR/RXR activation, FXR/RXR activation, and protein ubiquitination. At the end of the recovery phase, the 30 mg/kg bw FFC treated group altered pathways related to EIF2 signaling and lysine degradation.