The aim of this work was the analytical characterization of the β-orcinol depside, baeomycesic acid in lichens extracts. The extract of Thamnolia vermicularis var. subuliformis was analyzed by the two different methods, namely HPLC-UV and 1H NMR analysis. The results showed that baeomycesic acid was the most abundant depside in the lichens. These results could be of use for rapid identification of this metabolite in other lichen species. Besides baeomycesic acid, three depsides and one monocyclic phenolic compound were isolated from the lichen extract on the chromatographic column. The structure of baeomycesic acid was confirmed by HPLC-UV and spectroscopic methods. In addition, antioxidant and cytotoxic activities of baeomycesic acid were determined. The result of the testing showed that baeomycesic acid exhibited a moderate radical scavenging activity (IC50 = 602.10 ± 0.54 μg/mL) and good cytotoxic activity. This is the first report of detailed analytical characterization, isolation, as well as antioxidant and cytotoxic activities of baeomycesic acid from Thamnolia vermicularis. These results may be helpful in future industrial production of herbal medicines that include this important natural product.