Background: Multidrug-resistant Acinetobacter baumannii can cause complications in antibiotic therapy and increase the rate of morbidity and mortality in hospitalized patients. Patients with ventilator and burns are two specific groups at high risk for A. baumannii infections. This study aimed to determine antibiotic susceptibility patterns associated with biofilm production in A. baumannii and to assess its molecular epidemiology by random amplified polymorphic DNA polymerase chain reaction (RAPD PCR) in A. baumannii isolated from ventilator-associated pneumonia and burn wound colonization. Materials and Methods: In this study, 79 isolates of A. baumannii (32 ventilator-associated pneumonia [VAP] 47 burns) were collected in two teaching hospitals in Tehran, Iran, in 2018. Conventional biochemical and microbiological methods were used to identify bacteria. Antibiotic susceptibility was detected by disc diffusion methods according to the Clinical and Laboratory Standards Institute 2018. Tube test was examined for the detection of the biofilm formation rate in collected strains. The most prevalent carbapenemase genes were detected by PCR and molecular typing by RAPD PCR. Results: All of bacteria were extensively drug-resistant (XDR) except for two isolates. The results of tube test indicated that only 36% of XDR strains were in weak rate of biofilm formation group. Two major clonal genetic groups were found in VAP and burn strains. Oxa-23 was the most prevalent carbapenemase in collected A. baumannii. Conclusion: The presence of XDR strains of A. baumannii is considerable significant problem in hospitals. Further, similar genetic clonal identified in them indicated the nosocomial infection origin. Hence, these results are very important for control of nosocomial infection committee in health-care systems.