THE GROWING increase in the burden of hepatocellular carcinoma (HCC) across the world emphasizes the need for new modes of therapy. Therefore, Lepidium sativum has recently garnered increased attention for various medicinal uses. This study aimed to identify the chemical constituencies of garden cress seeds (methylene chloride and ethyl acetate extracts) and their impact on two HCC cell lines by measuring some apoptotic genes' expression. In addition, evaluation of their antimicrobial and antifungal activities. Regarding L. sativum's chemical composition, GC–MS analysis of the Methylene chloride extract led to identifying twenty-eight compounds, including hydrocarbons, terpenoids, and other volatile compounds. At the same time, the ethyl acetate extract identifies twelve compounds, including 3,4-dihydro-2H-1,5-(3"-T-butyl) benzodioxepine, cetane, linolenic acid, and oleic acid. However, both extracts revealed a weak antibacterial effect and no antifungal activity. The gene expression results revealed that all genes (TGFβ, SMAD3, BAX, and P53) showed upregulation trends in the HuH-7 and HEPG2 cells compared to the untreated control cells in both extracts of Lepidium sativum. Among Methylene chloride compounds, Molecular docking computations revealed higher binding affinities of compound 17 (Eugenol) against active EGFR (docking scores of −30.46 kcal/mol). Furthermore, the Pathway Enrichment Analysis results demonstrated that Neutrophil degranulation and Interleukin-4/Interleukin-13 signaling pathways were found to be the most significant pathways targeted by Eugenol, which may explain its potential as a therapeutic agent for cancer therapy.