Erythromycin (ERY) is a commonly used antibiotic that can be found in wastewater effluents globally. Due to the mechanisms by which they kill and prevent bacterial growth, antibiotics can have significant unwanted impacts on the fish gut microbiome. The overall objective of this project was to assess the effects of erythromycin and an antibiotic mixture on fish gut microbiomes. The project was split into two experiments to assess gut microbiome in response to exposure with ERY alone or in mixture with other common antibiotics. The objectives of experiment 1 were to understand uptake and depuration of ERY in juvenile rainbow trout (RBT) over a 7 d uptake followed by a 7 d depuration period using three concentrations of ERY. Furthermore, throughout the study changes in gut microbiome response were assessed. In experiment 2, a follow-up study was conducted using an identical experimental design to assess the impacts of an antibiotic-mixture (ERY, ampicillin, metronidazole, and ciprofloxacin at 100 µg/g each). Here, three matrices were analyzed, with gut collected for 16s metabarcoding, plasma for untargeted metabolomics, and brain for mRNA-seq analysis. ERY was depurated from the fish relatively quickly and gut microbiome dysbiosis was observed at 7 d after exposure, with a slight recovery after the 7 d depuration period. A greater number of plasma metabolites was dysregulated at 14 d compared to 7 d revealing temporality compared to gut microbiome dysbiosis. Furthermore, several transformation products of antibiotics and biomarker metabolites were observed in plasma due to antibiotic exposure. Brain transcriptome revealed only slight alterations due to antibiotic exposure. The results of these studies will help inform aquaculture practitioners and risk assessors when assessing the potential impacts of antibiotics in fish feed and the environment, with implications for host health. Overall design: Five RBT were exposed in each of six replicate 20-L tanks for each treatment in a dilutor system to regulate temperature (12°C) and light (14 h: 10 h light: dark). To maintain optimal water quality and consistency of the water microbiome, fish were housed under flow-through conditions with three complete renewals of cold, filtered facility water every 24 h. During experiments, water quality was assessed every 3 days. RBT were exposed via diet to two treatments of antibiotics, including 1,000 µg ERY/g and a mixture of antibiotics (ant-mix) containing nominal concentrations of 100 µg/g each of ampicillin, metronidazole, ciprofloxacin, and ERY, and one solvent control (MeOH). The 7-d exposure was followed by a 7-d depuration, during which food containing antibiotics was replaced with solvent control food. Brain mRNAseq was ran to assess effects of antibiotics on the transcriptome of the brain. Briefly, flash-frozen brain tissue was extracted for high-quality RNA using RNeasy Plus Universal Mini Kit with QIAzol Lysis Reagent (Qiagen, Germany). Two samples of whole brain collected from each tank after 7 d, were extracted and pooled at equimolar amounts with measurement using Qubit RNA HS Assay Kit (Thermo Fisher, USA). Extracted RNA was assessed for RNA integrity number (RIN) using Agilent RNA 6000 Nano kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Germany) with all values being = 6.5 with a mean ± standard deviation of 7.84 ± 0.875. Samples were then shipped on dry ice to Genome Quebec Innovation Centre for paired-end sequencing (2x100), using an Illumina NovaSeq 6000. Quality control of resulting sequence output was conducted using FastQC, and reads were assessed for quality, then trimmed to a minimum Phred score of 20 and a minimum length of 35 bases using Trimmomatic (Bolger et al., 2014). The abundance of transcripts was estimated using Salmon (version 1.10.2) with the reference RBT transcriptome USDA_OmykA_1.1 (GenBank Acc. no. GCA_013265735.3) (Patro et al., 2017) and mapped to genes using GenomicFeatures (version 1.52.1) (Lawrence et al., 2013) and corresponding gtf file (Oncorhynchus_mykiss.USDA_OmykA_1.1.110.gtf).