Rose Bengal (RB) is a potential photosuturing agent that may improve standard dermatologic surgical closure techniques. However, RB produces reactive oxygen species with photoactivation and its photomutagenic potential must be considered in clinical application. We investigated cytotoxicity, mutagenicity, and singlet oxygen (SO) production of RB on epithelial Chinese hamster ovary cell line. Cells were exposed to RB concentrations: 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%; irradiated for 400 s using a high-intensity visible wavelength lamp or maintained in the dark. Cell viability was assessed by XTT assay, mutagenicity by HPRT gene mutation assay, and SO production by Sensor Green reagent. RB > 0.001% was significantly cytotoxic. Viabilities were uninfluenced by ≤0.0001% RB controls, or 30-min incubation. 49% of irradiated cells died after 24-h in 0.0001% RB. At ≥0.001% RB, >90% of cells died. Irradiating 0.00001% - 0.001% RB increased SO; levels dropped significantly between 0.01% - 0.1%. Controls exhibited negligible SO production. HPRT suggested that RB was not mutagenic (0.0001%, 0.00001%); SO induction increased between 0.00001% - 0.001%, with reduced production at higher concentrations. Pilot studies suggested irradiated 0.0001% RB is mutagenic in vitro; current data suggest RB is not photomutagenic. The contribution of RB’s cytotoxicity on observed clinical improvement of scars and mutagenic potential remains unclear, necessitating further study.