Aim <p>Hepatocellular Carcinoma (HCC) is a fundamental health problem with an overall poor prognosis. <i>Jasonia montana (J. montana)</i> was evaluated for its biological activities against HCC cells (in vitro study).</p> Methods <p>The efficacy of <i>J. montana</i> was evaluated for its cytotoxic activity on HepG2 cells. Its impacts on cell cycle phases, apoptosis pathways, DNA damage, cellular oxidative stress, and inflammation were assessed. Additionally, the expression levels of <i>PTEN</i> and <i>SOX2</i> were also evaluated.</p> Results <p>The ethanol extract of <i>J. montana</i> produced a cytotoxic effect on HepG2 cells at (IC₅₀) of 24 μg/mL. There was a significant increase in the cell number in the 2n, G0/G1, and G2/M phases (p &lt; 0.001, p = 0.001, and p = 0.022; respectively), while a significant decrease in the cell number in the S phase (p &lt; 0.001) and 4n phase (p = 0.011) compared to the untreated HepG2 cells. Caspase-8 [2.6 ± 0.56 ng/mL versus 0.9 ± 0.06 ng/mL, p &lt; 0.001], Caspase-9 [11.9 ± 1.18 ng/mL versus 5.8 ± 0.85 ng/mL, p &lt; 0.001], and Caspase-3 [19.9 ± 1.01 ng/mL versus 4.0 ± 0.88 ng/mL, p &lt; 0.001] were significantly increased in HepG2 cells treated with <i>J. montana</i> compared to untreated control cells. There was a significant increase in the <i>PTEN</i> expression (FC = 3.04 ± 0.85, p = 0.030), and a significant decrease in <i>SOX2</i> expression (FC = 0.54 ± 0.02, p = 0.039). There was a significant decrease in IL-6 and TNF-α levels (P &lt; 0.001). Moreover, the total antioxidant (TA) and glutathione reductase (GSH) were significantly increased (P &lt; 0.001).</p> Conclusion <p><i>J. montana</i> could be a potentially useful chemotherapeutic agent against HCC cells.</p>