Background: Data on the prevalence of sick building syndrome (SBS) and its components in medical staff working at the Semnan University of Medical Sciences are limited. Objectives: The present study investigated the prevalence of SBS and its related factors in the medical staff in Semnan, Iran. Methods: A cross-sectional study was done on 300 medical staff in Kausar Hospital in Semnan, Iran, in 2020. A standard questionnaire was used to obtain information about SBS and indoor air quality. In order to measure environmental factors, random sampling was taken from different parts of the hospital, including building inspection, dust samples, and indoor and outdoor air measurements. The condition of physical factors, including temperature, humidity, and ventilation, was checked using relevant devices. The NIOSH 0800 method was used to measure the biological status of the working spaces in terms of fungal and bacterial contamination. Data analyses were performed using SPSS 26. Results: The mean age of the participants was 32.23 ± 5.34 years, of whom 81% were women. The prevalence of SBS was 65%, with the highest prevalence in the emergency ward (18%), followed by internal (16%), intensive care unit (13%), and cardiac care unit (12%). Sleep disorder (65%), tiredness (59%), feeling heavy (47%), headache (46%) and stress (45%) were the most common components of the SBS. There was a statistically significant relationship between the presence of harmful environmental factors, including noise, static electricity, unpleasant smell, and infection, and the prevalence of SBS (all P-values < 0.05). Conclusions: Considering the high prevalence of SBS in hospital employees, re-engineering the hospital environment and improving the air quality and ventilation systems may be useful in preventing SBS.