Abstract: Aquaculture is on the rise worldwide, and the use of antibiotics is fostering higher production intensity. However, recent findings suggest that the use of antibiotics comes at the price of increased antibiotic resistance. Yet, the effect of the oral administration of antibiotics on the mobility of microbial resistance genes in the fish gut is not well understood. In the present study, Piaractus mesopotamicus was used as a model to evaluate the effect of the antimicrobial florfenicol on the diversity of the gut microbiome as well as antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) using a metagenomic approach. The total relative abundance of ARGs and MGEs significantly increased during the antibiotic exposure. Additionally, phage integrases, transposases, and transposons flanking ARGs accumulated in the gut microbiome of P. mesopotamicus because of the antibiotic exposure. MGEs co-occurring with ARGs showed a significant positive correlation with the total ARGs found. Furthermore, shifts in the gut microbiome towards well-known putative pathogens such as Salmonella, Plesiomonas, and Citrobacter were observed following florfenicol treatment. Mainly Plesiomonas and Citrobacter harbored genes that code for multidrug and phenicol efflux pumps. Moreover, several genes related to RNA processing and modification, cell motility, SOS response, and extracellular structure were enriched due to the antibiotic application. The observed effects were visible during the complete application phase and disappeared at the post-exposure phase. Our findings suggest that the oral administration of antibiotics increases the potential for MGE-mediated exchange of ARGs in the gut of fish and could contribute to the enrichment and dispersion of ARGs in aquaculture systems. Importantly, this increase in the potential for ARGs exchange could be an effect of changes in community structure and/or ARG mobilization Ver menos