Lichens produce a large variety of secondary metabolites with diverse bioactivities, chemical structures, and physicochemical properties. For this reason, there is a growing interest in the use of lichen-derived bioactive molecules for drug discovery and development. Here, we report on the isolation, identification, and cytotoxic evaluation of gyrophoric acid (GA) from the lichen Umbilicaria muhlenbergii, a largely unexplored and scantly described lichen species. A simple purification protocol was developed for the fractionation of lichen crude extracts with silica gel column chromatography using solvents with changing polarity. GA was identified in one of the fractions with Fourier transform infrared spectroscopy (FTIR), ion trap mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (1H-NMR and 13C-NMR). The FTIR spectra demonstrated the presence of aromatic and ester functional groups C=C, C-H, and C=O bonds, with the most remarkable signals recorded at 1400 cm−1 for the aromatic region, at 1400 cm−1 for the CH3 groups, and at 1650 cm−1 for the carbonyl groups in GA. The MS spectra showed a molecular ion [M-1]− at (m/z) 467 with a molecular weight of 468.4 and the molecular formula C24H20O10. that correspond to GA. The 1H-NMR and 13C-NMR spectra verified the chemical shifts that are typical for GA. GA reduced the cell viability of breast cancer cells from the MCF-7 cell line by 98%, which is indicative of the strong cytotoxic properties of GA and its significant potential to serve as a potent anticancer drug. Keywords: lichen; Umbilicaria muhlenbergii; secondary metabolites; gyrophoric acid; breast cancer; cytotoxicity.