This study presents a comparative analysis of secondary metabolites and antioxidant, antimicrobial, and anticancer activities of acetone extracts obtained from the lichens Lepraria incana and Pertusaria amara. HPLC-UV analysis identified divaric acid, divaricatinic acid, norstictic acid, divaricatic acid and usnic acid in L. incana, and conprotocetraric acid, protocetraric acid, picrolichenic acid and atranorin in P. amara. Free radical scavenging capacity and reducing power assays were employed to assess the antioxidant activity of the extracts. The IC50 values in the free radical scavenging assay were 664.23 μg/mL for L. incana and 750.50 μg/mL for P. amara, while reducing power absorbances varied between 0.0875–0.2562 and 0.0336–0.2011, respectively. Total phenolic contents in L. incana and P. amara extracts were 40.81 and 33.67 μg PE/mg of extract, while total flavonoid contents were 24.74 and 23.61 μg RE/mg of extract, respectively. Antimicrobial activity, determined by the microdilution method, ranged from 156 to 20 × 103 μg/mL for L. incana and from 312 to 20 × 103 μg/mL for P. amara. Cytotoxicity was tested using the MTT method. Among the tested samples, the L. incana extract showed the strongest cytotoxic activity toward A549 cells, with an IC50 value of 47.53 μg/mL. Based on the results, the lichens examined demonstrate promise for future studies and potential development in biopharmaceutical applications. Keywords: Lepraria incana; Pertusaria amara; HPLC-UV; lichen metabolites; bioactivity.