Cannabis sativa L. is widely recognized as a medicinal plant. Its roots, which contain bioactive compounds with antioxidant and anti-inflammatory effects, have traditionally been used to treat several heath conditions, such as inflammation. C. sativa hairy root cultures produce higher levels of triterpenoid than wild-type roots, highlighting their potential as a sustainable source of bioactive metabolites. However, the pharmacological activities of C. sativa hairy root extracts (CAh), particularly their potential for cancer chemoprevention, remain poorly understood. This study aims to explore the chemopreventive potential of CAh against hepatocellular carcinoma (HCC). Network pharmacology and protein–protein interaction analyses, together with GO and KEGG enrichment analyses, identified several potential molecular targets associated with HCC, including SRC, PIK3CA, PTPN11, EGFR, JAK2, and ESR1. The effects of CAh on the expression of these candidates were subsequently evaluated in Huh-7 cells using RT-qPCR. Among the identified targets, CAh significantly downregulated ESR1 expression. Molecular docking and molecular dynamic simulations further revealed potential interactions between CAh-associated compounds and ESR1, providing structural insights into their possible modulation of ESR1. Collectively, these findings support further investigation of C. sativa hairy root-derived metabolites as potential candidates for HCC prevention.