Bacteria found in the operating room can lead to surgical site and hospital-acquired infections. This study was conducted in Imam Hussein Hospital in Nasiriyah to investigate the contamination levels in the operating rooms. The main goal of the research was to identify the bacteria responsible for contamination and the factors contributing to it. The study also aimed to map these microorganisms' distribution across different operating room areas and their antibiotic resistance pattern using microbiological standards. We gathered 1358 samples for analysis from surfaces and objects in the operating room. The results showed that 3.1% tested positive for bacteria, and 96.9% were negative cultures. Six types of pathogenic bacteria have been identified; Coagulase-negative staphylococci 14.3%, Staphylococcus aureus 11.9%, Pseudomonas aeruginosa 19.1%, E. coli 21.4%, Bacillus spp. 11.9%, and Enterobacter spp. 21.4%. We observed moderate to high resistance pattern to amoxicillin and ampicillin, Cefaclor, Cefuroxime, Cefadroxil, Erythromycin. The highest resistance pattern was detected in P. aeruginosa isolates followed by E. coli, it showed different resistance patterns to 14 antibiotics showing susceptibility to Amikacin only. Conclusion: the study at Imam Hussein Hospital found a generally low but notable level of bacterial contamination in orthopedic operating theaters, with specific pathogens posing risks to patients. These findings align with global data, underscoring common challenges in maintaining sterile surgical environments. Identifying contamination hotspots and patterns over time highlights the need for targeted interventions and continuous monitoring.