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Background: Nosocomial infections are associated with increased morbidity, mortality, and medical burdens. Pseudomonas aeruginosa and Acinetobacter baumannii are not-fermentative gram-negative bacteria that considered as the most important nosocomial infection. In the current study, we have aimed to evaluate the sensitivity of Acinetobacter baumannii and Pseudomonas aeruginosa microorganisms to the colistin antibiotic. Methods: In this descriptive cross-sectional study, patients admitted to the ICU ward of Firoozgar Hospital from July 2018 to March 2019 were evaluated, and 169 Patients infected with Acinetobacter baumannii, and Pseudomonas aeruginosa were included. Acinetobacter baumannii and Pseudomonas aeruginosa were isolated, and antibiotic sensitivity was determined by the disk diffusion method according to Clinical & Laboratory Standards Institute (CLSI) criteria. E test was also used to determine MIC-50 and MIC-90 of colistin. Results: Acinetobacter baumannii was around 8 times more frequent than Pseudomonas aeruginosa. Colistin resistance was detected in only 4(2.4%). The mean age of patients infected by Acinetobacter baumannii was significantly higher than those infected with Pseudomonas aeruginosa. Moreover, the mean time of the hospitalization period did not show any significant differences in the different groups. Conclusion: Our findings indicated that the majority of isolated Pseudomonas aeruginosa and Acinetobacter baumannii were sensitive to Colistin. Therefore, it could be effectively used for patients with a confirmed diagnosis of Pseudomonas aeruginosa and Acinetobacter baumannii.