The ambient air of hospitals contains a wide range of biological and chemical pollutants. Exposure to these indoor pollutants can be hazardous to the health of hospital staff. This study aims to evaluate the factors affecting indoor air quality and their effect on the respiratory health of staff members in a busy Iranian hospital. We surveyed 226 hospital staff as a case group and 222 office staff as a control group. All the subjects were asked to fill in a standard respiratory questionnaire. Pulmonary function parameters were simultaneously measured via a spirometry test. Environmental measurements of bio-aerosols, particulate matter, and volatile organic compounds in the hospital and offices were conducted. T-tests, chi-square tests, and multivariable logistic regressions were used to analyze the data. The concentration of selected air pollutants measured in the hospital wards was more than those in the administrative wards. Parameters of pulmonary functions were not statistically significant (p > 0.05) between the two groups. However, respiratory symptoms such as coughs, phlegm, phlegmatic coughs, and wheezing were more prevalent among the hospital staff. Laboratory staff members were more at risk of respiratory symptoms compared to other occupational groups in the hospital. The prevalence of sputum among nurses was significant, and the odds ratio for the presence of phlegm among nurses was 4.61 times greater than office staff (p = 0.002). The accumulation of indoor pollutants in the hospital environment revealed the failure of hospital ventilation systems. Hence, the design and implementation of an improved ventilation system in the studied hospital is recommended.