Intestinal microbiota contributes health of living organisms. Florfenicol is an approved antimicrobial (AM) prescribed for several bacterial fish diseases. The present study investigated the extent to which florfenicol modulates intestinal microbial populations of channel catfish (Ictalurus punctatus). Florfenicol was administered orally to catfish at a standard therapeutic dose (10–15 mg/kg of body weight for 10 days), and the intestinal contents were collected and the 16S rRNA was subjected to Illumina sequencing. Alpha diversity analysis indicated that florfenicol significantly decreased microbiota richness and diversity. Beta diversity reflected a clear separation had occurred between the florfenicol‐fed and control groups. Results indicated a significant increase in the abundance of phylum Proteobacteria (98.97% vs. 79.35% of the population) and decrease in phyla Firmicutes and Bacteroidetes (0.72% and 0.29% vs. 18.04% and 2.53%, respectively) in the florfenicol‐fed fish in comparison with the control fish. At the genus level, unclassified Enterobacteriaceae and Escherichia populations increased in the florfenicol group. In contrast, Plesiomonas, Aeromonas, Lactococcus, Clostridium sensu stricto, Romboutsia, Klebsiella, Turicibacter and Lactobacillus decreased in florfenicol‐fed fish in comparison with the control fish, indicating that intestinal microbiota of catfish was substantially modulated by florfenicol administration. Knowledge of changes in gut microbiota during medicated feed administration is important to improve fish performance and disease management and could enable the development of alternative therapeutic strategies.