Purpose: Commissioning must occur before an operating theatre (OT) is first used and after any substantial modifications that may affect airflow patterns in preexisting theatres (as part of a re-commissioning process). It is important that the infection control team (ICT) or microbiologist is involved at all stages from pre-design through to opening and that adequate time for commissioning is built in to the schedule, including an allowance of time for microbiological assessments. In the present study special emphasis laid on the process of commissioning OT with respect to microbiologist perspective. Material and Methods: The study was carried at the tertiary care hospital to commission newly built 6 OTs and one CATH lab. The sterilization of all 6 OTs and CATH lab was done in 3 cycles. First formaldehyde fumigation was done followed by two cycles of fogging using hydrogen peroxide. After fumigation, the sterile surface swabs were collected from representative areas from all OTs and CATH lab. The air sampling was done by settle plate method. Then all the swabs were processed in microbiology laboratory according to standard guidelines for aerobic, anaerobic and fungal cultures. Results: The OT and CATH lab surfaces were free from anaerobic contamination. The samples which were taken from door side AC duct in CATH lab grew Aspergillus niger. The settle plate did not yield any growth. Conclusion: It is necessary to perform Microbiology evaluation of the newly built OT before commissioning to ensure that ventilation system in the OT are functional, environmental parameters and the microbial load in the theater environment are at the acceptable level. It forms the integral part in the process of commissioning the OT.