Enterococcus faecium is a clinically significant nosocomial pathogen. The increasing prevalence of multidrug-resistant E. faecium (MDR E. faecium) poses a significant challenge to treatment and infection control efforts. This study investigates the MDR profiles of E. faecium clinical isolates, their phenotypic resistance characteristics, associated genetic determinants, and plasmid profiles. A total of 64 MDR E. faecium isolates from various clinical specimens were analyzed. Antibiotic susceptibility was assessed using the disk diffusion method, and minimum inhibitory concentrations (MICs) were determined. ... Resistance genes were identified using polymerase chain reaction (PCR), plasmid profiles were evaluated via gel electrophoresis, and conjugation assays were performed to evaluate transferability of resistance determinants. All isolates exhibited resistance to ampicillin, kanamycin, and erythromycin, with 96.9% exhibiting vancomycin resistance. High-level resistance to gentamicin (HLGR) and streptomycin (HLSR) was observed in 67.2% and 87.5% of isolates, respectively. PCR analysis confirmed the presence of vanA, vanB, erm(B), tetM, tetL, aac(6’)-aph(2”), ant(6)-Ia, and aphA3. Plasmid profiling revealed 1–10 plasmids per isolate, ranging from 2.08 to 56.15 kb. Detection of resistance genes and plasmid profiling support timely and informed interventions in both clinical management and antimicrobial strategies. These findings highlight the clinical value of molecular surveillance in managing MDR E. faecium.