The reduction in gut microbiota diversity is associated with a range of human diseases. Overuse of antibiotics has been associated with a diminished gut-microbial diversity in humans and may promote microbiota-associated negative effects to physical health, such as the metabolic syndrome-cluster of diseases and mental illnesses. There is a pressing need to deepen the understanding of the effects of antibiotics at the biochemical level. The current study investigated metabolic effects of two widely prescribed antibiotics–vancomycin and ciprofloxacin–on biofluids and brain tissue samples of NMRI female mice using a 1H nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling approach. While both antibiotics significantly affected the host metabolic signatures of urine and feces, only ciprofloxacin induced metabolic changes in plasma. Metabolic perturbations were pronounced 1 day post-treatment, reverting back to baseline at day 20 post-treatment. Both antibiotics induced changes in the choline metabolism, host-microbial cometabolites, short chain fatty acid production, and protein/purine degradation. The metabolic profiles of brain tissue aqueous extracts did not show any antibiotics-related changes by day 20 post-treatment. The data suggest that the metabolic disruptions in biofluids caused by antibiotics are reversed by day 20 post-treatment when compared to the pre-treatment profiles.