Background: Hospital-acquired infection is higher among healthcare workers (HCWs) than among other occupational groups, and the COVID-19 pandemic exacerbated its incidence. Aim: To assess the incidence of hospital-acquired infections among HCWs in, Cairo, Egypt, during the COVID-19 pandemic. Methods: This retrospective cohort study collected and evaluated data on hospital-acquired infections (excluding COVID-19) from 1660 nurses, physicians, technicians, and housekeepers in 6 hospitals in Cairo Governorate, Egypt, 1 year before and 1 year during the COVID-19 pandemic. The data were analysed using SPSS version 21.0 and descriptive and inferential statistics were used to test the magnitude and direction of relationships between the variables. Results: The rate of hospital-acquired infections was 3.1% before and 1.3% during the COVID-19 pandemic. There was a significantly lower risk of hospital-acquired infection among the HCWs during the pandemic than before it. Incidence was highest among housekeepers (10.6%), and physicians had the highest relative risk (4.33). Before the pandemic, pneumonia was the most common hospital-acquired infection (20.8%) among HCWs, while hepatitis C was the most common (8.3%) during the pandemic. The most significant predictors of hospital-acquired infection were COVID-19 (before and during the pandemic), working area and profession. Conclusion: There was a significant reduction in hospital-acquired infections among HCWs during the COVID-19 pandemic, attributed to increased adherence to infection prevention and control measures. Housekeepers, nurses and emergency department workers were at higher risk, indicating the need for further investigations among these groups of HCWs to understand the contributing factors and design targeted interventions to lower the risks and incidence.