Cannabis sativa L., specifically hemp, is a traditional herbaceous plant with industrial and medicinal uses. While much research has focused on cannabinoids and terpenes, the potential of hemp roots is less explored due to bioproduction challenges. Still, this material is rich in bioactive compounds and demonstrates promising anti-inflammatory, antimicrobial, and antioxidant properties. Biotechnological methods, such as hairy root cultures, enable the efficient production of specialized metabolites while avoiding the issues of outdoors cultures. Despite these benefits, the chemical diversity understanding of hemp hairy roots remains limited. In this study, we conducted an extensive NMR and LC/MS chemical profiling of hemp hairy roots to determine their chemical composition, revealing the presence of cannabisins for the first time. We then investigated the accumulation of cannabisins and triterpenes in both hemp hairy roots and hemp aeroponic roots. Our findings reveal that hairy roots produce 12 times more cannabisins and 6 times more triterpenes than aeroponic roots, respectively, in addition to yielding 3 times more biomass in bioreactors. Preliminary bioassays also suggest antioxidant and antifungal properties. This research underscores the potential of hemp hairy roots as a valuable source of specialized metabolites and calls for further exploration into their bioactive compounds and applications.