Background: Secondary metabolites are responsible for most of the biological activities of lichens. Many of these compounds exhibit significant antineoplastic activity. The aim of the present in vitro study was to evaluate the cytotoxic and genotoxic activities of organic extracts and purified barbatic acid from the lichen Cladonia salzmannii (Nyl.). Methods: The thallus of the lichen (22 g) was cleaned and dried with the solvents diethyl ether, chloroform and acetone. Organic extracts were obtained using the hot exhausted method in a Soxhlet apparatus. Barbatic acid (BAR) was purified from the ether extract (1.3 g). Chemical analysis of the organic extracts and purified BAR was performed using thin-layer chromatography. The purity of purified BAR was determined using high-performance liquid chromatography. The MTT method [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and cytokinesis-block proliferation index (CBPI) were used to determine the cytotoxic activity of the organic extracts and purified BAR. The micronucleus test and comet assay were used to determine genotoxic potential of the organic extracts and purified BAR. Dimethyl sulfoxide was used as the diluting solvent of the samples in all biological tests. Results: The IC50 results demonstrated significant cytotoxic potential of the ether extract (50 µg/mL) against cell lines NCI-H292 (IC50: 29.91 µg/mL), HEp-2 (IC50: 26.75 µg/mL) and HL-60 (IC50: 3.59 µg/mL) as well as the purified BAR (25 µg/mL) against cell lines HEp-2 (IC50: 15.79 µg/mL) and MCF-7 (IC50: 18.28 µg/mL). The CBPI demonstrated the cytotoxic activity of the purified BAR at all concentrations tested (5, 10, 20 and 40 µg/mL) and all organic extracts (50 µg/mL) against Ehrlich carcinoma cells. For sarcoma 180, only BAR purified at a concentration of 40 µg/mL and the ether and chloroform extracts (50 µg/mL) were considered cytotoxic. The micronucleus test revealed that the purified BAR at a concentration of 5 µg/mL had no genotoxic potential against either tumor cell line. Furthermore, the chloroform extract and purified BAR at a concentration of 10 µg/mL were not considered genotoxic for sarcoma 180. In the comet assay, all compounds tested induced DNA damage in both tumor lines. Conclusion: Based on the present results, organic extracts and purified barbatic acid from C. salzmannii exhibit cytotoxic and genotoxic activity against of the tumor cell lines tested.