Halogenoquinolones are potent and widely used antimicrobials blocking microbial DNA synthesis. However, they induce adverse photoresponses through the absorption of UV light, including phototoxicity and photocarcinogenicity. The phototoxic responses may be the result of photosensitization of singlet oxygen, production of free radicals and/or other reactive species resulting from photodehalogenation. Here, we report the use of laser scanning confocal microscopy to detect and to follow the fluorescence changes of one monohalogenated and three di-halogenated quinolones in live human epidermal keratinocyte cells during in situ irradiation by confocal laser in real time. Fluorescence image analysis and co-staining with the LysoTracker probe showed that lysosomes are a preferential site of drug localization and phototransformations. As the lysosomal environment is relatively acidic, we also determined how low pH may affect the dehalogenation and concomitant fluorescence. With continued UV irradiation, fluorescence increased in the photoproducts from BAY y3118 and clinafloxacin, whereas it decreased for lomefloxacin and moxifloxacin. Our images not only help to localize these phototoxic agents in the cell, but also provide means for dynamic monitoring of their phototransformations in the cellular environment.