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The aim of the present study was to examine the effect of gamma radiation on levofloxacin. Powder form of levofloxacin was subjected to different radiation doses (25, 50, 75, 100 and 125kGy) of Cobalt-60 source in a Gammacell-220 at a rate of 8.5 Gray/hr. The effect of radiation has been investigated with the aid of different spectroscopic techniques (UV-Vis, FT-IR), scanning electron microscopy (SEM), X-ray diffraction (XRD), and by antibacterial activities. UV data did not reveal significant changes in the structure of levofloxacin which is supported by scanning electron microscopy. However, X-rays diffraction shows a change in crystallinity of levofloxacin to an amorphous structure and this has been reflected on the morphology of this compound as indicated by SEM images. The antibacterial activities, on the other hand, reveal resistance of irradiated levofloxacin against bacteria, where some bacteria were highly affected by the irradiated drug. Similarly, FT-IR data show some changes in the functional groups principal absorption bands, in the IR spectrum, at frequencies 3286, 2846, 1716 and 1620 cm-1 for the O-H stretching band of quinolone, C-H stretching band, and C=O stretching band of carboxylic and pyridine. In addition, new peaks appeared which were not seen in the non-irradiated spectrum. In conclusion, some changes occurred in levofloxacin drug with the passage of radiation but the drug was chemically stable [ABSTRACT FROM AUTHOR]