Pseudomonas aeruginosa, a versatile Gram-negative bacterium, poses a serious threat due to its adeptness at developing resistance mechanisms. Metallo-beta-lactamases produced by P. aeruginosa are pivotal in conferring resistance to beta-lactam antibiotics, compromising treatment effectiveness. The bacterium’s proficiency in forming biofilms further enhances its persistence, especially in clinical settings. In this study conducted at Krishna Hospital and Medical Research Center, Karad, 205 clinical isolates of Pseudomonas aeruginosa were investigated for their metallo-beta-lactamase (MBL) production and biofilm-forming capabilities. The phenotypic detection of MBL production was carried out using the Imipenem-EDTA combination disc test, while biofilm formation was assessed through the microtiter plate technique. Genotypic detection involved conventional polymerase chain reaction targeting MBL and biofilm genes. Results showed that 28.29% of isolates produced MBL phenotypically, with 16.59% possessing MBL genes, notably the bla_NDM-1 gene in 15.12% of isolates. Regarding biofilm production, 85.85% of isolates were biofilm producers, and biofilm-encoding genes were present in 80.49% of isolates. The most frequently encoded genes were algD (79.51%), pelF (58.0%), and pslD (46.34%). Comparing phenotypic and genotypic methods for MBL detection, a statistically significant average agreement was observed. While there was an increasing trend in biofilm genotypic positive patterns with stronger biofilm phenotypic patterns, the correlation was not statistically significant. The study concludes that every resistant clinical isolate should be screened for MBL and biofilm production using simple phenotypic tests, with confirmation by PCR if possible. This comprehensive analysis provides insights into the prevalence and genetic basis of MBL and biofilm in P. aeruginosa clinical isolates, contributing to strategies for combating antibiotic resistance.