The COVID-19 pandemic changed the healthcare environment and raised concerns about the impact on nurse staffing and healthcare-associated infections (HAIs), but evidence on this impact is lacking. This study evaluated changes in nurse staffing levels in South Korea and their impact on healthcare-associated COVID-19 infection (HA COVID-19). A total of 67,154 cases were included, and survival analysis was performed using a Cox proportional hazards model to assess the association between nurse staffing and HA COVID-19. Among patients admitted to intensive care units and general wards, 11.7% and 15.1%, respectively, contracted COVID-19 in the hospital after admission. Survival analysis showed that HA COVID-19 increased as the nurse-to-patient ratio increased in general wards. Additionally, compared with medical institutions that do not operate fixed night shift nurses, HA COVID-19 increased as the proportion of night nurses increased, but it decreased to less than 10%. This study shows that securing nurses and operating an appropriate proportion of night nursing staff is important for infection control. To prepare for the spread of infectious diseases and implement infection prevention activities, it is necessary to secure an appropriate level of nursing staff; maintaining an appropriate proportion of night shift nurses will help reduce HAIs.