The diversity of microbial species in the gut has a strong influence on health and development of the host. Further, there are indications that the variation in expression of gut bacterial metabolic enzymes is less diverse than the taxonomic profile, underlying the importance of microbiome functionality, particularly from a toxicological perspective. To address these relationships, the gut bacterial composition of Wistar rats was altered by a 28 day oral treatment with the antibiotics tobramycin or colistin sulfate. On the basis of 16S marker gene sequencing data, tobramycin was found to cause a strong reduction in the diversity and relative abundance of the microbiome, whereas colistin sulfate had only a marginal impact. Associated plasma and fecal metabolomes were characterized by targeted mass spectrometry-based profiling. The fecal metabolome of tobramycin-treated animals had a high number of significant alterations in metabolite levels compared to controls, particularly in amino acids, lipids, bile acids (BAs), carbohydrates, and energy metabolites. The accumulation of primary BAs and significant reduction of secondary BAs in the feces indicated that the microbial alterations induced by tobramycin inhibit bacterial deconjugation reactions. The plasma metabolome showed less, but still many alterations in the same metabolite groups, including reductions in indole derivatives and hippuric acid, and furthermore, despite marginal effects of colistin sulfate treatment, there were nonetheless systemic alterations also in BAs. Aside from these treatment-based differences, we also uncovered interindividual differences particularly centering on the loss of Verrucomicrobiaceae in the microbiome, but with no apparent associated metabolite alterations. Finally, by comparing the data set from this study with metabolome alterations in the MetaMapTox database, key metabolite alterations were identified as plasma biomarkers indicative of altered gut microbiomes resulting from a wide activity spectrum of antibiotics.