The investigation evaluates the expertise of healthcare personnel regarding the contamination of hospital ambient air with bacteria. The objective of the investigation is to establish a correlation between participant knowledge and both independent and dependent factors. Cross-sectional analytical research was conducted by three institutions in Najaf City from mid-December 2023 to May 10, 2024. Data from 515 health professionals who completed a random self-reporting questionnaire were analyzed using a statistical instrument. The primary findings suggest that the majority of participants are women, with 51.8% of them falling within the 20–29 age range. Of these women, half have bachelor's degrees (54.6%), and 44.5% have less than five years of experience. 27.0% of the professions were in a laboratory setting, and 30.1% had received training. The data indicates that 13.4% of individuals possessed a high level of general knowledge. We discovered substantial correlations between all sociodemographic factors and knowledge, with the exception of sex, which did not exhibit a significant correlation with knowledge. The survey revealed that the majority of health professionals (68.0%) possessed only middling levels of knowledge. Consequently, it is imperative that local authorities provide ongoing training to health personnel regarding bacterial indoor air pollution in order to elevate these levels. We arrived at the conclusion that there is a substantial correlation between knowledge and training programs. Additionally, we identified a statistically significant correlation between the educational attainment of the preponderance of youthful healthcare professionals.