Introduction: Microbiological contamination of various operation theatres of tertiary care hospitals by potential pathogens has led to the rise in incidence of health care associated infections. The aim of the present study is to assess, compare and correlate microbial contamination levels in operation theatres using both an active and passive sampling methods. Material &Methods: A prospective study conducted in the Department of Microbiology, Modern Government Maternity Hospital, Hyderabad, Telangana, for a period of 1 month from July 1st to August 1st, 2023. Sampling was performed in 7 Operation theatres both at rest (before beginning the surgical activity) and during surgery. Air from one operating room per day was sampled by both Active and Passive methods at the same time, at rest and during surgery for one month. In Active sampling method, microbiological air sampler is used which physically draws a known volume of air over a particle collection device media i.e., Sheep blood agar and the number of colonies grown on the media is measured in CFU/m3of air. In passive sampling method, Sheep blood agar plates were placed in OTs, according to 1/1/1 rule and results are expressed in CFU/m2 /hour. Results: The mean number of colonies is 17.4 CFU/ m3 and 63.1 CFU/m2 /hour at rest; 42.4 CFU/ m3 and 186.69 CFU/m2 /hour during surgery for active and passive air sampling methods respectively. Statistical analysis (spearmen’s rank correlation coefficient) confirmed that the two methods are correlated with each other in a comparable way with the quality of air. Conclusion: The microbiological quality of the air in operating theatres is a significant parameter to control healthcare associated infections, and regular microbial monitoring can represent a useful tool to assess environmental quality and to identify critical situations which require corrective intervention. Hence, it is possible to conclude that both the methods are correlated with each other, but passive method can be used for general monitoring of air contamination, such as routine surveillance programs.