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The aim of this study was to identify some of the secondary metabolites present in acetonic, methanolic, and hexanic extracts of lichen <i>Xanthoparmelia stenophylla</i> and to examine their antioxidant, antimicrobial, and cytotoxic activity. Compounds of the depsid structure of lecanoric acid, obtusic acid, and atranorin as well as usnic acid with a dibenzofuran structure were identified in the extracts by HPLC. The acetone extract was shown to have the highest total phenolic (167.03 ± 1.12 mg GAE/g) and total flavonoid content (178.84 ± 0.93 mg QE/g) as well as the best antioxidant activity (DPPH IC₅₀ = 81.22 ± 0.54). However, the antimicrobial and antibiofilm tests showed the best activity of hexanic extract, especially against strains of <i>B. cereus</i>, <i>B. subtilis,</i> and <i>S. aureus</i> (MIC < 0.08, and 0.3125 mg/mL, respectively). Additionally, by using the MTT method, the acetonic extract was reported to exhibit a strong cytotoxic effect on the HeLa and HCT-116 cell lines, especially after 72 h (IC₅₀ = 21.17 ± 1.85 and IC₅₀ = 21.48 ± 3.55, respectively). The promising antioxidant, antimicrobial, and cytotoxic effects of <i>Xanthoparmelia stenophylla</i> extracts shown in the current study should be further investigated in vivo and under clinical conditions.