Microbiological air contamination in operation theatres (OTs) and labor rooms (LRs) is a major risk factor for surgical site infection. Routine monitoring in vulnerable areas such as OTs and LRs should always be performed as part of infection control to evaluate the contamination by microorganisms and monitor for the presence of nosocomial agents. The present study is aimed to isolate and identify various pathogens in a hospital environment. The research was performed in the Bacteriology lab, Microbiology Department, Dr.Patnam Mahender Reddy Institute of Medical Sciences, Chevella, Ranga Reddy district, Hyderabad, India between November 2017 and November 2020 for a period of 3 years. Surface swabbing and settle plate techniques were two sampling techniques used in the analysis. A total of 3492 samples were collected from various hospital surface sites and 5 OTs and LR sites and equipment via the swab technique, while a Petri plate gravitational settling (passive) sampling method was selected for the collection of air samples and all these samples were properly transferred to the microbiology laboratory and processed by standard microbiological protocols. A total of 3492 surface swabs were taken from 5 OTs, LR sites and equipment from the hospital. Out of these 294 (8.42%) were culture positive and 3198 (91.58%) were culture negative. Among 294 microbial isolates, the highest number was reported from Bacillus spp. 212 (72.11%) and least number was from Pseudomonas spp. 6 (2.04%). Through air sampling methods, bacterial isolates were isolated from OTs and LR of various clinical departments and it was found that the highest bacterial count was reported from general surgery (677 CFU/m3) followed by orthopaedics (585 CFU/m3). Most of the microbial isolates isolated from OTs and LR of clinical departments found be species belonging to Bacillus spp. and Coagulase-negative Staphylococci (CoNS). In developing countries, routine indoor air quality management in healthcare facilities needs to be constantly monitored and appropriate measures are taken to detect and prevent acquired infections in hospitals. Settle plate methods for air and surface swabbing, also in resource-limited settings, are very useful, simple and cost-effective techniques for OT and LR monitoring.