Abstract Glansreginin A has been reported to be an indicator of the quality of walnuts ( Juglans spp.). However, bioactive properties of glansreginin A have not been adequately explored. In the present study, we quantified concentrations of glansreginin A in black walnuts ( Juglans nigra ) using high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) and performed an array of in vitro bioassays to characterize biological activities (e.g., antibacterial, antioxidant, anticancer capacities) of this compound. Results from HPLC–MS/MS analysis indicated that glansreginin A was presented in all 12 black cultivars examined and its contents were variable among black walnut cultivars, ranged from 6.8 mg/kg (Jackson) to 47.0 mg/kg (Hay). Glansreginin A possessed moderate antibacterial activities against Gram-positive pathogens ( Staphylococcus aureus and Bacillus anthracis ). This compound exhibited no antioxidant activities, did not induce the activity of antioxidant response element signaling pathways, and exerted no antiproliferative effects on tumorigenic alveolar epithelial cells and non-tumorigenic lung fibroblast cells.