Employees working in various sections of the hospital are inevitably exposed to toxic gases. Chronic exposure to these gases increases the risk of occupational diseases. Therefore, this study aims to evaluate the impact of chemical exposure on pulmonary and blood parameters. This cross-sectional study was conducted in 2024 in a hospital located in the Tehran with a sample size of 110 participants. In this study, OSHA 103, NIOSH 1501, OSHA 101, NIOSH 7901, and NIOSH 3500 were used to determine the exposure levels of isoflurane, BTEX, formaldehyde, nitric acid, and chlorine compounds, respectively. Respiratory symptom history was obtained using the American Thoracic Society Questionnaire (ATSQ). In addition, lung function was determined by spirometry tests, while blood samples were taken to analyze the participants’ blood factors. The results of the present study showed that the average occupational exposure of staff in the operating room and pathology laboratory during 8 hours was 0.16 for formaldehyde and 6.1 for isoflurane, both of which were above the ACGIH recommended occupational exposure level (OEL). All other chemicals were reported to be lower than the recommended limits. Analysis of spirometry test results and blood factors in both the control and case groups revealed a correlation between the prevalence of respiratory symptoms and elevated liver enzymes in employees exposed to formaldehyde and isoflurane. The results indicated a significantly higher prevalence of respiratory symptoms and liver enzyme disorders in individuals exposed to isoflurane and formaldehyde compared to the control group. Furthermore, a significant difference was found in pulmonary function tests and liver enzyme levels in the blood of subjects exposed to the control group. It is recommended that people working in the operating room and pathology laboratory must apply the engineering and management methods of occupational health and safety and use personal protective equipment due to exposure to impermissible levels of isoflurane and formaldehyde.