BACKGROUND: A growing concern among nurses is that they have become extremely stressed and anxious, for many reasons. Intestinal flora has become a widely research topic. Stool samples were sequenced to explore the relationship between intestinal flora and the stressors of nurses. METHODS: Paediatric nurses in the Third Xiangya Hospital were the research group, and internal medicine nurses were the control group. The “Chinese Nurse Stressor Scale” (CNSS) was used for general investigation and stress-related assessment. Six patients in the internal medicine group and 7 in the paediatric group underwent DNA sequencing of their intestinal flora. The gut microbiome was profiled by 16S rRNA sequencing. RESULTS: The work stressors of internal medicine and paediatric nurses included nursing specialty and work (10.94±3.17 vs 12.25±2.78), work environment and resources (4.91±1.63 vs 5.66±2.03), patient care (15.86±4.30 vs 18.95±5.12) and management and interpersonal relationships (8.83±4.94 vs 10.86±5.01). The abundance of Clostridia in the internal medicine group was higher, while the abundance of Bacteroidia in the paediatric group was higher. The abundances of γ-Proteobacteria and β-Proteobacteria also showed obvious differences between the two groups. CONCLUSIONS: The changes in the intestinal flora of nurses were correlated with their stress levels, which were especially notable for Bacteroidia, Clostridia, γ-Proteobacteria and β-Proteobacteria.