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Background: Multidrug-resistant bacteria (MDR) often contaminate hospital environment and cause serious illnesses. Quorum Sensing (QS) regulates a variety of downstream cellular processes, including antibiotics resistance mechanisms and biofilm formation, and causes harm to the host. This study investigates antibacterial susceptibility and biofilm formation of pathogenic bacteria in hospital environment. Methods: Hundred bacterial isolates were collected from various environments in the Medical City hospital. The antimicrobial susceptibility technique was evaluated through disk diffusion method. Next, biofilms formation was detected by the microliter plate assay. Finally, PCR was used to analyze the frequency of QS system genes. Results: Current findings showed that the predominant isolates were Acinetobacter baumannii (34%), Escherichia coli (30%), Pseudomonas aeruginosa (19%), and Klebsiella pneumonia (17%). In general, significant resistance was found related to trimethoprim (88%), Augmentin (88%), and cefotaxime (72%). Among all isolates, 62% of sensitivity was related to ciprofloxacin. Biofilm had been formed by 39% of isolates. PCR results showed that the frequency of lasI and rhlI gene was 70% and 61%, respectively. Conclusion: Current findings revealed that the hospital environment is a potential reservoir of MDR gram-negative pathogenic bacteria. Thus, we suggest that the health policymakers in Iraq must critically apply the guidelines and recommendations for monitoring the environments in the health sector. [ABSTRACT FROM AUTHOR]