Lichens are symbiotic organisms consisting of fungi and algae with notable bioactive potential. This study optimized extraction conditions and quantified total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity of Usnea species using Response Surface Methodology (RSM)-Design Expert 13. The Usnea species was collected from three host plant species: Hagenia abyssinica (Kosso), Hypericum revolutum (Amija), and Erica arborea (Wuchena), from the Ras Dashen Mountain region within Simien Mountains National Park (SMNP), northern Gondar, Ethiopia. Methanol concentration (40-100% v/v) and extraction time (60-180 min) were evaluated as independent variables, with optimum yield achieved at 100% methanol and at 115, 130, and 120 minutes for Usnea species collected from Hagenia abyssinica, Hypericum revolutum, and Erica arborea. A UV-Vis spectrophotometer was used to measure the total phenolic and flavonoid contents and the antioxidant activity of the samples. The optimum values of TPC, TFC, and antioxidant activity obtained were 161.06 ± 0.73 mg GAE/100g DW, 413.68 ± 1.82 mg CE/100g DW, and 73.39 ± 0.98 % inhibition (Hagenia abyssinica);111.14 ± 1.24 mg GAE/100g DW, 256.14 ± 1.61 mg CE/100g DW, and 53.52 ± 1.1 % inhibition (Hypericum revolutum); and 123.44 ± 1.07 mg GAE/100 g DW, 429.12 ± 2.19 mg CE/100g DW, and 71.1 ± 0.96 % inhibition (Erica arborea) respectively. Beyond phenolic and antioxidant characterization, lipid content determination revealed that Usnea species exhibited varying lipid levels: 16.34% (w/w) in the sample collected from Hagenia abyssinica, 29.92% (w/w) from Hypericum revolutum, and 15.9% (w/w) from Erica arborea. These findings provide a comprehensive biochemical characterization of Usnea species, demonstrating their potential as a source of antioxidants and nutritionally relevant lipids, and supporting further exploration of their applications in nutritional and pharmaceutical development