Antibiotics have been shown to have a major impact on the composition and metabolism of gut microbiota, while unpredictable stress has been shown to have major influences on gut microbiota homeostasis. However, the impact of combination antibiotics (e.g. metronidazole and ciprofloxacin) on gut microbiota dysbiosis that is induced by stress remains unclear. Here, chronic unpredictable mild stress (CUMS) was used to simulate unpredictable stress, and Sprague-Dawley rats received antibiotics (metronidazole and ciprofloxacin) after CUMS. The weights and coefficients of the thymus, liver and spleen were analysed. 16S rRNA gene sequencing was performed to determine the gut microbiota in the colon, cecum and ileum. We found that antibiotics decreased the CUMS-induced increases in spleen coefficients. Short-term antibiotic exposure significantly increased the cecum and ileum microbiota richness and significantly increased the ileum microbiota diversity after CUMS. Long-term antibiotic exposure significantly decreased the colon microbiota diversity and significantly increased the ileum microbiota richness after CUMS. The impacts of antibiotic exposure on the microbiota compositions at the phylum and genus levels in different gut segments were different after CUMS. However, it is worth noting that the most differentially abundant bacteria in the colon, cecum and ileum were all Verrucomicrobia under long-term antibiotic exposure. Antibiotic exposure significantly increased the relative abundance of Lactobacillus in the colon and ileum and significantly increased the relative abundance of Akkermansia in the colon and cecum after CUMS. In conclusion, this study showed that metronidazole and ciprofloxacin differentially affected the CUMS-induced changes in the microbiota of the colon, cecum and ileum.