Ciprofloxacin is a widely used fluoroquinolone drug with broad spectrum antibacterial activities. Clinical experience has shown incidences of adverse effects related to skin, hepatic, central nervous system, gastrointestinal and phototoxicity. India is a tropical country and sunlight is abundant throughout the day. In this scenario exposure to ambient levels of ultraviolet radiation (UV-R) in sunlight may lead to harmful effects in ciprofloxacin users. Phototoxicity assessment of ciprofloxacin was studied by two mouse fibroblast cell lines L-929 and NIH-3T3. Generation of reactive oxygen species (ROS) like singlet oxygen (1O2), superoxide anion radical (O2ḃ−) and hydroxyl radical (ḃOH) was studied under the exposure of ambient intensities of UV-A (1.14, 1.6 and 2.2 mW cm−2), UV-B (0.6, 0.9 and 1.2 mW cm−2) and sunlight (60 min). The drug was generating 1O2, O2ḃ− and ḃOH in a concentration and dose-dependent manner. Sodium azide (NaN3) and 1,4-diazabicyclo 2-2-2-octane (DABCO) inhibited the generation of 1O2. Superoxide dismutase (SOD) inhibited 90–95% O2ḃ− generation. The drug (5–40 μg mL−1) was responsible for linoleic acid peroxidation. Quenching study of linoleic acid peroxidation with SOD (25 and 50 U mL−1) confirms the involvement of ROS in drug-induced lipid peroxidation. The generation of ḃOH radical was further confirmed by using specific quenchers of ḃOH such as mannitol (0.5 m) and sodium benzoate (0.5 m). 2′-deoxyguanosine (2′-dGuO) assay and linoleic acid peroxidation showed that ROS were mainly responsible for ciprofloxacin-sensitized photo-degradation of guanine base. L-929 cell line showed 29%, 34% and 54% reduced cell viability at higher drug concentration (300 μg mL−1) under UV-A, UV-B and sunlight, respectively. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in NIH-3T3 cell line at higher drug concentration (300 μg mL−1) showed a decrease in cell viability by 54%, 56% and 59% under UV-A, UV-B and sunlight, respectively. Results of neutral red uptake assay (NRU) in L-929 cell line were in accordance with MTT assay. The NIH-3T3 cell line showed a higher photosensitizing potential than L-929. The phototoxicity end point shows a time- and concentration-dependent statistically significant (P < 0.001) damage. Ciprofloxacin produced ROS by Type I and Type II photodynamic reactions, interacted with nucleic acid moiety and inhibited cell viability. Further, UV-induced photo-peroxidation of linoleic acid accorded the involvement of ROS in the manifestation of drug phototoxicity. Appearance of ciprofloxacin-induced phototoxicity at the ambient level of sunlight is a real risk for the people of India and for those of other tropical countries. We suggest that sunlight exposure should be avoided (especially peak hours) during ciprofloxacin treatment.