Background: Hospital acquired infections caused by multi-drug resistant organisms are a significant health risk in Trauma and Surgical Intensive Care Units (TICU and SICU), leading to increased morbidity, mortality, and healthcare costs. The rise of multidrug-resistant (MDR) organisms exacerbates these challenges, making effective infection control and antimicrobial stewardship critical for improving patient outcomes. Objective: This study aimed to investigate the microbial epidemiology and prevalence of multidrug-resistant (MDR) organisms in the Trauma and Surgical Intensive Care Units (TICU and SICU) at Beni-Suef University Hospital, with the goal of guiding targeted infection control strategies and enhancing antimicrobial stewardship efforts. Methods: A cross-sectional study was conducted over a seven-month period from September 2023 to February 2024, involving 140 patients admitted to the TICU and SICU. Clinical specimens, including blood, sputum, and urine samples, were collected and analyzed using standard microbiological methods. Microbial identification was performed through traditional culturing techniques, microscopic examination, and biochemical tests, with further verification using automated identification systems. Antimicrobial susceptibility testing was conducted following the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Results: A total of 117 isolates were obtained from 140 patients, with 88.89% identified as multidrug-resistant organisms (MDROs). A significant statistical difference was observed in the occurrence frequencies of these organisms between TICU and SICU (p < 0.05). The predominant bacterial pathogens included Klebsiella pneumoniae (54 isolates; 46.15% of all isolates), Pseudomonas aeruginosa (14 isolates; 11.97%), and Escherichia coli (8 isolates; 6.84%). Fungal pathogens, including Candida albicans and Aspergillus spp., were also identified. Notably, MDR strains were prevalent in Acinetobacter baumannii (5.13% of all isolates), Pseudomonas aeruginosa (11.97%), and Klebsiella pneumoniae (30.77%), highlighting the escalating threat of antibiotic resistance. Conclusion: The study underscores a consistent microbial burden across TICU and SICU, with variations in the frequency of specific organisms. These findings highlight the urgent need for targeted infection control measures and robust antimicrobial stewardship programs to combat the rising threat of MDR organisms. Future research should focus on developing strategies to mitigate the impact of these pathogens on patient outcomes.