The rise in multidrug-resistant pathogens poses a formidable challenge in treating hospital-acquired infections, particularly those caused by . Biofilm formation is a critical factor contributing to antibiotic resistance, enhancing bacterial adherence and persistence. strains vary in virulence factors, influencing their pathogenicity and resistance profiles. This study aimed to comprehensively analyze virulence factors, antibiotic resistance patterns, and biofilm formation in clinical isolates of from Hamadan hospitals. Moreover, the study explored the molecular epidemiological relationships among isolates using multilocus sequence typing (MLST) to uncover the genetic diversity associated with resistance and virulence. Between December 2022 and April 2024, 402 isolates were collected from clinical samples, including urine, tracheal aspirates, blood, wounds, and abscesses, in teaching hospitals in Hamadan. Initial culturing was performed on blood agar and MacConkey agar, and isolates were identified using biochemical tests. Antimicrobial susceptibility testing followed CLSI, employing the Kirby-Bauer disk diffusion method with 10 antibiotics. Biofilm formation was assessed using the microtiter plate method, and virulence genes were detected by PCR. MLST analysis was conducted on 10 selected isolates based on their virulence gene profiles and resistance patterns. Of the 456 clinical isolates analyzed, 402 (88.15%) were identified as , predominantly isolated from tracheal samples (251/402, 62.44%), followed by urine (105/402, 26.12%), blood (30/402, 7.46%), wounds (15/402, 3.73%), and abscesses (1/402, 0.25%). Antibiotic resistance rates revealed high resistance to cefepime (356/402, 88.55%), imipenem (345/402, 85.82%), and ceftazidime (305/402, 75.87%), while resistance to amikacin (165/402, 41.04%) and piperacillin-tazobactam (75/402, 18.65%) was comparatively lower. Biofilm formation varied among the isolates, with 17/402 (4.22%) forming strong biofilms, 104/402 (25.87%) moderate biofilms, 180/402 (44.78%) weak biofilms, and 101/402 (25.12%) showing no biofilm production. Virulence gene analysis indicated high prevalence rates for (396/402, 98.50%), (351/402, 87.31%), and (402/402, 100%), while genes like (151/402, 37.56%) and (136/402, 33.83%) were less common, and and were absent. MLST analysis of 10 selected isolates identified sequence types ST147 (5/10, 50%), ST11 (3/10, 30%), and ST15 (2/10, 20%). demonstrates notable biofilm-associated antibiotic resistance, supported by a significant association with XDR strains, along with a diverse array of virulence gene profiles. The study underscores the importance of understanding molecular epidemiology for effective management of hospital infections, emphasizing the need for targeted surveillance and infection control measures.