Background: According to ethnobotanical data, Elaeagnus angustifolia fruit has wound healing activity, anti-inflammatory effect and antifebrile prosperities. Objectives: This study was performed as to the best of our knowledge; there has been no scientific report on the characterization of antimicrobial effect of E. angustifolia extract. Materials and Methods: An aqueous extract of Elaeagnus angustifolia was prepared and antimicrobial activity tests were performed on various target cultures. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the extract was done using the broth dilution technique. To characterize the extract, shelf life, thermal and pH stability, effects of detergents such as Tween 80, Tween 20, Triton X100, toluene and enzymes on the antimicrobial activity of Elaeagnus angustifolia extract, were examined. Results: The MIC values ranged from 7.5 to 0.1 mg/mL, showing maximum activity (1.62 mg/mL) against E. coli. Similarly, the MBC of the extract against E. coli was 1.62 mg/mL. Antimicrobial activity of the extract was relatively stable when kept in the refrigerator for 60 days. The antimicrobial activity of Elaeagnus angustifolia extract was absolutely stable at temperatures up to 700° C. After exposure of the Elaeagnus angustifolia extract to different pH solutions in the range of 4-10, almost 100% residual activity was found against E. coli at pH 4, 5, 6, and 7. Treatment of the extract with detergents, lipase and lysozyme eliminated its antimicrobial activity. Conclusions: Our study gives an indication of the presence of promising antimicrobial compounds and provides basic information about the nature of the Elaeagnus angustifolia extract. Future studies should elucidate the components responsible for antimicrobial activity of these extracts against target cultures.