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Introduction: Pseudomonas aeruginosa is one of the most prevalent nosocomial pathogens that cause a life-threatening infection. One of the important characteristics of P. aeruginosa is biofilm formation and the most studied bacterium related to biofilm formation so far. The biofilm formation and beta-lactamases production synergistically contribute to the extensive dissemination of multi-drug resistant strains. Aim: The present study was conducted to identify, biofilm-producing isolates of P. aeruginosa along with their antibiotic resistance pattern and ESBL and MBL production and to analyze their antibiogram. Materials and methods: Various clinical specimens were collected and totally 82 clinical isolates of P. aeruginosa were included in this study. Biofilm producing isolates were identified by the tube adherence method. Results: Among the total, 22 [26.83%] isolates were biofilm producers and the maximum number was obtained from blood [100%], followed by ETT [75%], and Drain [66.67%]. Biofilm producing isolates were showing more resistance in comparison to non-biofilm producers. All isolates of P. aeruginosa were sensitive to colistin and polymyxin B. Association of ESBL production and biofilm formation found to be statistically significant [p < 0.002], which was a contrast to association of MBL production and biofilm formation. Conclusion: High-level resistance to antimicrobial agents is a characteristic feature of infection caused by biofilm and lead to chronic infections. Knowledge about these biofilm-producing isolates is important in the clinical setting to eradicate these chronic and life-threating infections.