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Background Pseudomonas aeruginosa is one of the most important opportunistic pathogens responsible for hospital-acquired infections. Its remarkable ability to produce biofilms significantly contributes to antimicrobial resistance, persistent infections, prolonged hospitalization, and increased mortality. Biofilm-associated genes such as algD, pelA, pslA, and lasB play a vital role in biofilm formation and bacterial virulence. Understanding the association between biofilm production, biofilm-forming genes, and antimicrobial resistance is essential for improving infection control strategies and therapeutic outcomes. Objectives 1. To isolate and identify Pseudomonas aeruginosa from various clinical specimens. 2. To detect biofilm production using the Tissue Culture Plate (TCP) method. 3. To identify biofilm-forming genes using Polymerase Chain Reaction (PCR). 4. To evaluate antimicrobial susceptibility patterns. 5. To determine the association between biofilm formation, biofilm genes, and multidrug resistance. Materials and Methods A hospital-based cross-sectional study was conducted over 12 months in the Department of Microbiology of a tertiary care hospital in Indore, Madhya Pradesh. A total of 180 non-duplicate clinical isolates of P. aeruginosa were recovered from urine, pus, wound swabs, respiratory secretions, blood, and other clinical specimens. Identification was performed using standard microbiological methods and biochemical tests. Antimicrobial susceptibility testing was carried out according to CLSI guidelines using the Kirby-Bauer disc diffusion method. Biofilm production was assessed by the microtiter plate assay, and PCR was used to detect algD, pelA, pslA, and lasB genes. Results Among the 180 isolates, 112 (62.2%) were biofilm producers, including 41 (22.8%) strong, 46 (25.6%) moderate, and 25 (13.9%) weak producers. The prevalence of biofilm-associated genes was algD (90.2%), pslA (84.8%), pelA (79.5%), and lasB (92.0%) among biofilm-producing isolates. Biofilm producers demonstrated significantly higher resistance to ceftazidime (74.1%), ciprofloxacin (68.8%), meropenem (57.1%), imipenem (54.5%), and gentamicin (63.4%) compared with non-biofilm producers (p<0.001). Multidrug resistance was observed in 61.6% of biofilm producers compared with 23.5% of non-biofilm producers. Conclusion Biofilm formation is highly prevalent among clinical isolates of Pseudomonas aeruginosa and is strongly associated with multidrug resistance. Detection of biofilm-associated genes provides valuable information regarding bacterial virulence and antimicrobial resistance, emphasizing the need for routine surveillance and effective antibiotic stewardship in tertiary care hospitals.