Background: Nosocomial sepsis is a leading cause of morbidity and mortality worldwide. Understanding the antibiotic susceptibility patterns of pathogens specific to each geographic region is essential for effective patient management. Methods: This cross-sectional study aimed to investigate the microorganisms causing nosocomial sepsis and their antibiotic sensitivity in patients admitted to three referral hospitals in Isfahan, Iran. Clinical information and blood culture results were obtained from hospital records. Results from patients without signs of sepsis, as well as contaminants and community-acquired isolates, were excluded. Stratification was conducted based on sex and age (< 20 and > 20 years) categories. Results: In this study, 267 patients with hospital-acquired sepsis were identified, with gram-negative bacteria accounting for 77.2% of infections. The most common pathogens included Klebsiella spp. (35.2%), Acinetobacter spp. (21.7%), Enterococcus spp. (13.5%), Escherichiacoli (8.6%), Pseudomonasaeruginosa (8.6%), and Staphylococcusaureus (8.2%). The highest resistance among gram-negative bacteria was observed with cefepime (80.3%), ceftazidime (76.5%), cefotaxime/ceftriaxone (70.0%), trimethoprim-sulfamethoxazole (68.6%), ciprofloxacin (68.2%), meropenem (60.0%), and amikacin (50.3%). All gram-negative isolates were sensitive to colistin. Among gram-positive bacteria, the highest resistance was to ciprofloxacin (77.3%), ampicillin (75.7%), clindamycin (74.4%), vancomycin (64.9%), and gentamicin (30.0%). All gram-positive isolates in this study were sensitive to linezolid. Conclusions: The study indicates a high level of resistance among bacterial agents causing nosocomial sepsis in the studied area. Consequently, treatment may necessitate the use of last-line antibiotics, such as linezolid and colistin.