Background: Acinetobacter is a critical nosocomial pathogen responsible for various infections. It represents global threat due to high antibiotic resistance, including to last-resort options, and possesses multiple virulence factors that lead to significant morbidity and mortality rates. Objectives: This study aims to investigate the association between different antibiotic susceptibility patterns and the presence of phospholipase D (pld) and serum resistance (traT) virulence genes in Acinetobacter isolated from clinical samples in ICUs. Methods: Clinical specimens of hospital-acquired infections were collected from ICUs at Tanta University Hospitals. Acinetobacter isolates were identified using conventional methods, and their antibiotic susceptibility was assessed through disk diffusion. Colistin susceptibility was tested by broth macrodilution, and virulence genes (pld and traT) were detected using conventional PCR. Results: Out of 135 clinical samples, (20.7%) were identified as Acinetobacter, with 96.4% classified as multi-drug resistant (MDR). The isolates showed high resistance to cefotaxime (100%), piperacillin/tazobactam (92.9%), and ceftazidime (92.9%), while low resistance was noted for tetracycline (28.6%) and colistin (10.7%). All isolates (100%) carried the pld gene, and (82.1%) had the traT gene. Isolates with both virulence genes exhibited significantly higher resistance rates against imipenem (82.6%), ciprofloxacin (87%), and aminoglycosides (73.9%), along with absolute resistance (100%) to cefotaxime and ceftriaxone. MDR levels were notably high in both groups of virulence-associated gene carriage (95.7% for group 1 and 100% for group 2). Conclusions: There is high prevalence of antimicrobial resistance of Acinetobacter isolates among medical ICU patients with a high proportion of virulence-associated genes.