BACKGROUND: A series of breast cancer cases were recently reported in a tertiary university hospital in South Korea. Nurses are generally exposed to risk factors for breast cancer such as night shift work, antineoplastic agents, and job strain. However, the epidemiological evidence of excess incidence among nurses remains lacking. This study aims to investigate the excess incidence of breast cancer among nurses in a tertiary university hospital and provide epidemiological evidence of occupational risk factors. METHODS: A retrospective cohort was developed using personnel records of female workers in the nursing department who worked from January 2011 to June 2021 in a tertiary university hospital in South Korea. Sick leave records were used to identify cases of breast cancer. The standardized incidence ratio of breast cancer among nurses was compared to the general population. RESULTS: A total of 5,509 nurses were followed up for 30,404 person-years, and 26 breast cancer cases were identified. This study revealed a significantly increased breast cancer incidence among all included nurses, with a standardized incidence ratio of 1.65 (95% confidence interval [CI]: 1.08-2.41), compared to the general population. Workers, who handle antineoplastic agents in their representative department and current and/or former department, had significantly elevated breast cancer standardized incidence ratios of 2.73 (95% CI: 1.008-5.94) and 3.39 (95% CI: 1.46-6.68), respectively. CONCLUSIONS: This study provides significant evidence of increased breast cancer risk among nursing staff in a hospital setting, particularly those who handle antineoplastic drugs. Measures that reduce exposure to risk factors should be implemented, especially anticancer drugs, to protect healthcare professionals. Further research at a national level that focuses on healthcare workers is necessary to validate breast cancer incidence and its contributing factors.