: Occupational burnout and fatigue are critical issues affecting the health and performance of healthcare workers (HCWs) globally. These outcomes are often driven by complex and overlapping work-related stressors, which remain insufficiently understood in combination. : To investigate the associations of multiple work-related stressors with occupational burnout and fatigue, and to identify distinct stress patterns and critical stressors among HCWs. : A cross-sectional survey was conducted using a self-administered electronic questionnaire among 2695 HCWs in Shanghai, China. Validated questionnaire scales were used to assess work-related stress (self-developed occupational stress scale for medical staff, CSSM), occupational burnout (Maslach Burnout Inventory-General Survey, MBI-GS), and fatigue (Fatigue Scale-14, FS-14). Latent profile analysis (LPA) was employed to identify distinct work-related stress patterns. Generalized linear models (GLMs) were used to explore the associations between individual stressors, stress patterns, and occupational burnout and fatigue. Additionally, weighted quantile sum (WQS) models were utilized to evaluate the combined effects of multiple stressors and identify the main contributors. : In this study, 77.0% and 71.2% of participants were classified as experiencing occupational burnout and fatigue, respectively. A strained doctor-patient relationship was the highest-rated work-related stressor. All work-related stressors, including career development, interpersonal relationships, work-life imbalance, physical environment, doctor-patient relationship, social environment, and workload, were significantly associated with burnout (β: 0.4440.956, < 0.001) and fatigue (β: 1.3843.404, < 0.001). WQS assigned higher weights to career development and workload for burnout, and to workload and work-life imbalance for fatigue. LPA identified two distinct occupational stress patterns. HCWs characterized by higher stress levels in physical environment, career development, workload, and interpersonal relationships exhibited significantly higher burnout scores (β = 0.325, 95% CI: 0.122, 0.528), particularly in the reduced personal accomplishment (PA) dimension (β = 1.003, 95% CI: 0.746, 1.259). : This study highlighted the high prevalence of occupational burnout and fatigue among HCWs in Shanghai, China. Occupational stressors were associated with both burnout and fatigue, with higher workload, work-life imbalance, and poorer career development showing particularly significant contributions. These findings emphasized the urgent need for targeted interventions, including workload management, career development programs, and mental health support, to reduce occupational stress and mitigate its adverse effects on HCWs.