Background <p>Lichens produce several secondary metabolites with significant biological and pharmacological activities, among which usnic acid (UA) is particularly notable. This study aimed to investigate the cytotoxic activity, effects on cell migration and invasion, and the mechanisms underlying UA-induced cell death in the human hepatocarcinoma cell line Hep3B.</p> Methods <p>Hep3B cells were treated with UA for 24 and 48&#xa0;h to evaluate its cytotoxicity. Gene expression levels associated with apoptosis and cell migration were analyzed. Functional assays, including scratch-wound and Matrigel invasion assays, were employed to assess migration and invasion. Additionally, molecular docking studies were conducted to elucidate the interactions between UA and potential protein targets.</p> Results <p>UA demonstrated significant cytotoxic effects on Hep3B cells, with an IC₅₀ of 19.677&#xa0;µM at 48&#xa0;h. UA treatment induced apoptosis and effectively suppressed cell migration and invasion. Molecular docking analyses revealed strong binding interactions between UA and key target proteins, as evidenced by negative binding energy values for all complexes.</p> Conclusions <p>These findings indicate that UA exerts cytotoxic effects on Hep3B cells by inducing apoptosis and inhibiting migration and invasion. The molecular docking results further suggest UA as a potential lead compound for anticancer drug development. Further investigations focusing on its molecular targets and derivatives are warranted to explore its full therapeutic potential.</p>