We studied the nature and antimicrobial activity of ozonolysis transformation products (OTPs) of levofloxacin (LEV), a frequently detected fluoroquinolone antimicrobial in environmental waters. Two bioassays, the Kirby-Bauer test and the broth microdilution assay, were used to measure changes in the antimicrobial activity of solutions at low LEV to O3 molar ratios (2:1, 2:3 and 1:3) compared to solutions without added O3 (LEV:O3 1:0). The Kirby-Bauer test was not sensitive enough to detect significant differences in the growth inhibition zones in samples LEV:O3 2:1 and LEV:O3 1:0; however, the broth microdilution assay showed that bacterial growth inhibition was significantly lower (P<0.001) in the solutions exposed to O3. Loss of antimicrobial activity in LEV:O3 2:1 solutions of (48±16)% was in agreement with the concentration decrease of LEV of (36±3)% in those same samples. A method of identification of OTPs using XCMS Online was applied to LEV:O3 2:1 and 1:0 samples and indicated the presence of an OTP of LEV of formula C18H20O5N3F, which was identified as LEV-N-oxide. The molecular structure of this compound was partially confirmed by tandem mass spectrometry experiments. This study showed that even at sub-optimal ozone doses, OTPs of higher antimicrobial activity than LEV were not formed.