Background: Numerous studies have demonstrated that daphnetin (DAP) has anti-inflammatory, antioxidant, antibacterial, antitumor, anti-arthritic, and other extensive pharmacological effects. However, its genotoxicity has rarely been examined. Materials and Methods: The safety of DAP was evaluated by an acute toxicity test using a Salmonella typhimurium/mammalian microsomal enzyme assay (Ames test), bone marrow micronucleus test, acute skin allergy test, and local mucosal irritation test. Results: Mice received a dose of 100 mg/kg body weight through lavage, which is more than 175 times the whole-body effective dose and were continuously observed for 14 d; mice showed no signs of poisoning. In the Ames test, each dose of DAP resulted in numbers of revertant colonies that were <2 two times the number of mutation colonies in the negative control group, indicating a negative result. The micronucleus rate of each DAP dose group was not significantly different from that of the negative control group (P > 0.05) but was significantly different from that of the cyclophosphamide (CTX)-positive control group (P < 0.05). Rabbit skin showed no stimulation reaction to DAP or 10% dimethyl sulfoxide (DMSO), including no erythema, edema, or sensitization phenomena. Rabbits were generally in good condition after dosing the oral mucosa with DAP, and no oral mucosal erythema, erosion, ulcers, or other irritation reactions were observed. Conclusions: Within the range of tested doses, DAP did not cause any observed toxic effects, mutagenic effects, sensitization, or stimulation.