Florfenicol is a broad-spectrum antibiotic approved by the US Food and Drug Administration (FDA) to treat both systemic and external bacterial infections in food fish. The objective of this study was to evaluate the effect of Florfenicol (FFC)-medicated feed on the gut microbiota of zebrafish to determine i) if the therapeutic dose of FFC-medicated feed induces dysbiosis, and ii) if fish with altered gut microbiota were more susceptible to subsequent infection by the common opportunistic fish pathogen Aeromonas hydrophila. We used 16S rRNA gene sequencing to analyze the gut microbiota of FFC-medicated feed-treated zebrafish, and our results found that FFC-medicated feed induced disruption of the gut microbiota. Dysbiosis was observed in all treated groups with a significant increase in bacterial diversity, and it was characterized by a remarked bloom of Proteobacteria and a drastic decline of Mycoplasma and Cetobacterium in treated animals without noticeable clinical signs or mortalities. In addition, the increase of Proteobacteria wasn´t significantly reduced after the recommended 15-day antibiotic-withdrawal period, and the zebrafish treated with FFC-medicated feed exhibited a significantly higher mortality rate when they were subsequently challenged with A. hydrophila compared to the regular feed groups. Interestingly, the most dramatic changes in the gut microbiome composition occurred at the transition time between the late stage of the medicated treatment and the beginning of the withdrawal period, instead of the time during the Aeromonas infection. To conclude, the administration of FFC-medicated feed at the recommended dose induced gut dysbiosis in zebrafish, and it did not recover to the baseline after the recommended withdrawal period. Our findings suggest that the use of antibiotics in fish elicits a similar response to those previously described in mammals and possibly makes the host more susceptible to subsequent infections of opportunistic pathogens. This study using a controlled model system suggests that antibiotics in aquaculture may have long-term effects on the general wellbeing of the fish.