The fluoroquinolone Rufloxacin (RFX) is active as specific inhibitor of bacterial gyrase. The adverse effects of the photosensitization induced by fluoroquinolones are well known. A predominant type II photosensitizing activity of Rufloxacin has already been demonstrated on simpler models (free nucleosides, calf thymus DNA), whereas a cooperative mechanism was corroborated on more complex ones (plasmid and fibroblast). The purpose of this study is to examine the drug photocytoxicity in another complex cellular model, a wild-type eukaryotic fast-growing microorganism whose cultivation is cheap and easily managed, Saccharomyces cerevisiae. This work represents the first report of the potential photogenotoxicity of Rufloxacin. Particular emphasis was given to DNA modifications caused in yeast by the formation of Rufloxacin photomediated toxic species, such as hydrogen peroxide and formaldehyde. Drug phototoxicity on yeast was evaluated by measuring DNA fragmentation (single/double strand breaks) using single cell gel electrophoresis assay and 8-OH-dGuo, a DNA photooxidation biomarker, by HPLC-ECD. Cellular sensitivity was also assessed by cell viability test. The extra- and intracellular RFX concentration (as well as its main photoproduct) was verified by HPLC-MS, whereas the cytotoxic species were evaluated by colorimetric assays. The results confirm the phototoxicity of Rufloxacin on yeast cell and are in agreement with those previously obtained with human fibroblast and with simpler models used recently, and provide a clear link between DNA photosensitization and overall phototoxicity.