Background: Increased resistance to antibiotics among Acinetobacter baumannii (A. baumannii) isolates is a rising problem, and new alternatives should be provided to overcome this problem. Toxin-Antitoxin system (TA) is considered a promising essential target for antimicrobial drugs. Objective: To detect the prevalence of toxin-antitoxin system in A. baumannii isolated from patients at Zagazig University Hospitals. Methodology: Following isolation, oxidase test and API20NE were used to identify A. baumannii, antibiotic susceptibility testing was performed to all isolates obtained, and amplification and screening of functional mazEF, relBE and higBA toxin-antitoxin genes were done by PCR and RT-PCR, respectively. Results: Out of 252 clinical specimens collected, 27(10.7%) were A. baumannii; 13 (15.3%) isolates were isolated from endotracheal aspirates, 4 (13.3%) from sputum samples, 7 (9.2%) from urine, 2 (4.5%) from pus, and 1 (14.3%) from blood. Most of the isolates were multidrug resistant, and the highest susceptibility was to meropenem (66.7 %) followed by imipenem (63%). Regarding PCR results, 22 isolates (81.5%) had relBE gene, 17 (62.9%)had mazEF gene, and 8(29.6%) had higBA gene. In the RT-PCR results, all genes were functional in all isolates. Conclusion: TA system genes are prevalent among A. baumannii isolates, in particular; relBE and mazEF genes and they are functional.