Lichenized fungi are a source of secondary metabolites with multiple biological potential. The objective of the study was to determine the chemical composition, antioxidant, and anti-inflammatory activity of the hydroalcoholic extract of the Antarctic lichen Leptogium puberulum through metabolomic, in vitro, and in silico analyses. Seventeen compounds were tentatively identified using UHPLC-ESI-QToF-MS. The phenolic composition yielded 6.356 mg GAE/g, and antioxidant activity assays showed IC50 values for DPPH• and ABTS•+ of 1187.149 and 207.00 µg/mL, respectively, along with 15.845 µmol Trolox/g for ORAC and 21.925 µmol Trolox/g for FRAP. The in silico evaluation was performed using OSIRIS Data Warrior, ProTox 3.0, and SwissTargetPrediction, identifying 9,10,12,13,14-pentahydroxytetracosanoic acid (PHTA), 9,10,12,13-tetrahydroxytricosanoic acid (THTA), 9,10,12,13-tetrahydroxyheneicosanoic acid (THHA), and 9,10,12,13-tetrahydroxydocosanoic acid (THDA) as the most promising compounds. These metabolites showed favorable pharmacokinetic properties, with no anticipated toxicological risks. Subsequently, their affinity for the cyclooxygenase-2 (COX-2) enzyme was evaluated by molecular docking with AutoDock Vina software version 1.2.3, and the most stable protein–ligand complexes were analyzed to characterize key interactions within the active site and subjected to molecular dynamics simulations with YASARA software version 19.1.27 for 100 ns. Overall, these results indicate that selected metabolites from L. puberulum may act as potential COX-2 inhibitors, supporting their relevance as lichen-derived anti-inflammatory agents and warranting further pharmacological investigation. Keywords: Antarctica; extract; bioactive compounds; metabolomics; biological potential; pharmacokinetic properties; molecular docking; COX-2 inhibition.