In this study, a solution containing the fluoroquinolones (FQs) ciprofloxacin, lomefloxacin, and ofloxacin (antimicrobial agents) was subjected to photocatalytic oxidation under UVA irradiation, employing the commercial titanium dioxide as catalyst. On-line solid phase extraction coupled to ultra-high-performance liquid chromatography–mass spectroscopy was used to pre-concentrate and quantify the analytes. The process provided an almost 95% degradation efficiency for all the FQs. The TiO2 PC500 (100% anatase) was more efficient than TiO2 P25 (80% anatase) for FQs degradation. The matrix effect on the efficiency of the process was evaluated by ultrapure water – UW, simulated water – SW, bottled water –BW, and public drinking tap water – TW. Simulated water showed lower interference, compared to drinking water and bottled mineral water, due to the lower concentrations of hydroxyl radical scavengers. The assessment of the residual antimicrobial activity in the solution, when using 50 mg L−1 PC500 or 100 mg L−1 P25, showed reductions of biological activity (after 120 min of reaction) of 92.4% and 95.4% for Escherichia coli, and 78.1% and 84.2% for Bacillus subtilis, respectively. It shows that the photocatalytic oxidation process was able to not only degrade the FQs but also deactivate its biological activity in the resultant solution.