Enterobacteriaceae are bacteria found in the digestive system, known for causing infections and displaying antibiotic resistance. The World Health Organization (WHO) identifies strains resistant to third-generation cephalosporins and carbapenems as priority pathogens. This study, conducted at Avicenne Military Hospital of Marrakech, aimed to assess the epidemiological and antibiotic resistance profiles of Enterobacteriaceae over a two-year period (May 2020 - April 2022) to inform updated antibiotic use recommendations. This retrospective study analyzed 1,626 positive bacterial isolates from various hospital departments, including medical, surgical, intensive care, emergency units, and external samples. Of these, 1,163 (71.5%) were Enterobacteriaceae, predominantly Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae, primarily isolated from urine, pus, and blood cultures. Antibiotic resistance was assessed, revealing that 614 (52.8%) of Enterobacteriaceae were extended-spectrum beta-lactamase producers (ESBL-E), and 115 (9.9%) were carbapenemase-producing Enterobacteriaceae (CPE). The study’s findings align with existing literature, showing a higher prevalence of Enterobacteriaceae in males and frequent isolation of Escherichia coli. The rise in multidrug-resistant Enterobacteriaceae, particularly ESBL-E and CPE, poses risks such as therapeutic failure, increased morbidity and mortality, and significant economic impacts. To address this, the study emphasizes the importance of early detection, infection control measures, responsible antibiotic prescribing, and collaborative efforts across hospital departments to reduce the spread of resistant strains.