Recently antibiotics wastewater in environment has induced the increment of bacterial resistance. This paper is to investigate the gamma radiolysis of wastewater containing 10 μg/mL levofloxacin lactate (LVF). It has been found that the antibiotic was removed more than 99% with 1 kGy under air while the G-value decreased with the dose increment. Five main degraded products (,m/z, 346, 330, 318, 302, 274) and the most probable radiolysis pathway were identified by liquid chromatography-mass spectrometry (LC-MS). Compared with the degradation under N,2, the radiolytical mechanism was suggested. In the active assay, 2 μg/mL was the inhibitory concentration. Compared with the concentration of 4 μg/mL, the minimal inhibitory concentration (MIC), degraded products analysis can be suggested that the byproduct m/z 346 might have the anti- E. coli activity. It has been shown that 3 kGy is the appropriate dose for the radiolytical treatment of LVF. To sum up, the gamma radiation technique is an effective method for decomposing antibiotics, and it is necessary to take the activity of degraded products into consideration.