Industrial hemp roots are rich in various non-cannabinoid types of phytochemicals including phenolics, alkaloids, phytosterols, and triterpenoids. However, the biological activities of these compounds are not well studied, which hindered the development of hemp roots-based functional ingredients. Herein, we aimed to evaluate the anti-inflammatory effects of a panel of non-cannabinoid phytochemicals isolated from a hemp roots extract. Representative chemotypes of compounds included cannflavins A and B (flavonoids), dadahol A (lignan), indole-3-carboxaldehyde (alkaloid), stigmast-4-en-3-one (phytosterol), and friedelin (triterpenoid). The anti-inflammatory effects of these were evaluated in a cell-based model using differentiated human monocytes (THP-1 cells) challenged with an inflammation inducer (i.e., lipopolysaccharide; LPS). These hemp roots phytochemicals (at 10 µM) reduced the pro-inflammatory cytokine interleukin 1β in THP-1 cells exposed to LPS by 11.5%-61.3%. Among the test samples, cannflavins A and B showed the most promising anti-inflammatory activity with an IC50 value of 6.7 and 8.2 µM, respectively. Furthermore, cannflavins A and B (at 10 µM) showed an inhibitory effect in the activation of NLRP3 Inflammasome in THP-1 cells exposed to LPS and nigericin by 33.6 and 29.1%, respectively. Moreover, the mechanisms of cannflavins’ anti-inflammatory effects were studied by network pharmacological analysis and molecular biology approaches, which suggest that cannflavins A and B may mediate molecular targets such as the caspases family. Findings from our study shed light on the biological activities of non-cannabinoid compounds from industrial hemp roots, which provided useful information for the further development of hemp root extracts as promising active nutraceutical and/or cosmeceutical ingredients.