Multiple drug resistance (MDR) in Acinetobacter baumannii strains is a rapidly rising phenomenon worldwide. Different mechanisms are responsible for this resistance and efflux pumps are among them. In A. baumannii resistance resulted from efflux pumps are mainly in Major facilitator super family (MFS) and Resistance nodulation cell division (RND) families. Therefore, this study has been conducted to study in vitro evaluation of MFS efflux pumps among multidrug resistant A. baumannii isolated from patients hospitalized in intensive care unit (ICU). In this cross-sectional study, 100 MDR isolates were selected from a total of 200 clinical A. baumannii isolated from ICU; Multiplex- RT- PCR methods was used to in vitro evaluation of MFS efflux pumps among multidrug resistant A. baumannii isolated from patients hospitalized in ICU. According to the susceptibility test, among 200 A. baumannii isolates, the prevalence rate of MDR, non MDR and extremely drug resistance (XDR) were 56%, 34% and 10% respectively. Abaye 0369 gene expression was observed in MDR and non-MDR isolates in presence of tetracycline which is an indication of its role in antibiotic resistance against tetracycline. In confirming the role of MFS transporters such as abaye 0369 in tetracycline resistance, functional studies should be designed. For assessing the effect of the expression of other studied MFS transporters in tetracycline resistance, their expression rate should be determined using Real Time PCR.