This study aimed to explore Evernia prunastri, lichen potential as a source of bioactive flavonoids and natural precursors of vitamin D2. Extracts obtained by maceration and Soxhlet were subjected to phytochemical analysis, revealing a high content of total phenolics (25.5 mg GAE/100 g) and total flavonoids (0.285 mg QE/g). Antioxidant, anti-inflammatory and antibacterial activities were evaluated using in-vitro assays. Antioxidant potency, measured by DPPH assay, revealed IC₅₀ of 0.049 mg/mL, indicating notable activity. Inhibition of protein denaturation (indicator of anti-inflammatory activity), showed IC₅₀ of 388.52 μg/mL. The extracts also had moderate antibacterial activity against Escherichia coli and Pseudomonas aeruginosa. Finally, the detection of ergosterol a fungal sterol characteristic of lichens and a precursor of vitamin D₂ (ergocalciferol) by gas chromatography-mass spectrometry (GC/MS) confirmed the nutraceutical activity of this specie. This plant-based form of vitamin D is distinct from animal-derived vitamin D₃ (cholecalciferol), derived from 7-dehydrocholesterol. These results showed Evernia prunastri as a promising source of bioactive compounds with therapeutic and nutritional potential.