Fatigue has gradually become an important factor affecting the health and work quality of healthcare workers. However, the occurrence of fatigue and related factors in the context of heavy workloads and strained doctor-patient relationships have not been adequately studied in Northeast China. The current study aims to assess the situation of fatigue among healthcare workers in tertiary general hospitals in Northeast China, and to further analyze the influencing factors of fatigue among healthcare workers (doctors and nurses as a whole), doctors and nurses from various perspectives. A cross-sectional survey was conducted among healthcare workers who met the inclusion criteria in two tertiary general hospitals in Dalian, Liaoning Province, Northeast China. The survey data collected in the structured questionnaire include fatigue, general demographic characteristics, work situation, perceived stress, insomnia symptoms, workplace violence, anxiety symptoms, depressive symptoms, family function, and doctor-patient relationship. A binary logistic regression model was applied to analyze the independent influencing factors of fatigue. A total of 1004 healthcare workers were included in this study (503 doctors and 501 nurses). The prevalence of fatigue was 53.9% for healthcare workers, with rates of 52.3% for doctors, and 55.5% for nurses. Binary logistic regression analysis showed that health status, perceived stress, insomnia symptoms, anxiety symptoms, and doctor-patient relationship were influencing factors of fatigue among healthcare workers, doctors, and nurses. Chronic pain was an influencing factor for fatigue among healthcare workers and nurses. Depressive symptoms were an influencing factor for fatigue for healthcare workers and doctors. Night shift frequency was an influencing factor for fatigue for nurses. In Northeast China, tertiary general hospitals have a relatively high prevalence of fatigue among healthcare workers, especially among nurses. Fatigue was influenced by various factors such as health status, perceived stress, insomnia symptoms, anxiety symptoms, and doctor-patient relationships. It is necessary to raise awareness of fatigue among healthcare workers, particularly healthcare workers with risk factors, and to develop targeted and effective intervention measures aimed at reducing fatigue.