Background: Health workers (HWs) are exposed to diverse cleaning agents in large hospitals. This study determined the prevalence of work-related symptoms, allergic sensitization, and lung function abnormalities in HWs of two tertiary hospitals in Southern Africa. Methods: A cross-sectional study of 699 HWs (South Africa: SAH, n = 346; Tanzania: TAH, n = 353) was conducted. Health outcomes were assessed using a standardized ECRHS questionnaire, immunological tests (specific IgE antibody to common aeroallergens and to occupational allergens: natural rubber latex [NRL] Hev b5 and Hev b6.02, chlorhexidine, and ortho-phthalaldehyde [OPA]), spirometry [pre-and postbronchodilator], methacholine challenge, and fractional exhaled nitric oxide (FeNO).Results: A large proportion of participants (78%) were women. Median age was 42 years, with 76% nurses, 12% cleaners, and 5% administrative workers. Current smoking was more common in SAHWs (12%) than TAHWs (1%). The overall prevalence of doctor-diagnosed asthma was 7%. Atopy was present in 43% of HWs, while 4% were sensitized to OPA, 2% to NRL, and 1% to chlorhexidine. Prevalence of work-related ocular-nasal symptoms (16%) was higher than skin (12%) and chest (7%) symptoms. TAHWs had significantly lower mean lung volumes, higher degrees of significant airflow obstruction and impaired lung function. The prevalence of bronchial hyperresponsiveness in SAHWs (14%) was high. Overall, 23% of HWs had abnormal FeNO; 6% having high (>50 ppb) levels. FeNO was positively associated with sensitization to occupational allergens, primarily OPA and NRL.Conclusions: HWs from both hospitals had similar prevalence of work-related respiratory symptoms. Sensitization to OPA and NRL appears to be contributing to allergic airway inflammation in these HWs.