Klebsiella pneumoniae is an important opportunistic pathogen associated with a wide range of hospital-acquired infections. One of the major virulence factors of this bacterium is its ability to form biofilms, which enhance bacterial persistence and resistance to environmental stress. The present study aimed to investigate the phenotypic and molecular detection of biofilm formation in Klebsiella pneumoniae isolates obtained from different clinical cases in Al-Hillah city, Iraq. A total of 200 clinical samples, including sputum and urine specimens, were collected from hospitalized patients. The samples were cultured on Blood agar, MacConkey agar, and EMB agar for bacterial isolation and identification. Out of the total samples, 20 isolates were identified as Klebsiella pneumoniae using conventional biochemical tests and the VITEK automated identification system. The phenotypic detection of biofilm formation was performed using the microtiter plate (MTP) method with crystal violet staining. The results showed that 17 isolates (85%) were strong biofilm producers, while 3 isolates (15%) showed moderate biofilm formation. Molecular detection of biofilm-related genes was carried out using PCR. The results demonstrated that the treC gene (302 bp) was present in all isolates (100%), whereas the cpsD gene (386 bp) was detected in 9 isolates (45%). These findings indicate that Klebsiella pneumoniae isolates possess a strong ability to produce biofilms and harbor important genes associated with biofilm formation.