Traditional/herbal medicine has gained increasing interests recently, especially in Asian countries such as Vietnam, due to its diverse therapeutic actions. In the treasure of Vietnamese medicinal plants, one of the potential herbs is the roots of Sophora flavescens Ait. (SF, “Kho sam” in Vietnamese). However, limited information has been reported on the Vietnamese SF compositions and their respective alkaloids’ anti-acetylcholinesterase action. Thus, this study investigated the extractions, isolations, identifications, and in-vitro antioxidant, cytotoxicity, and acetylcholinesterase inhibitory activities, of the SF root extracts and their purified alkaloid compounds. To this end, four pure compounds were successfully isolated, purity-tested by HPLC, and structurally identified by spectroscopic techniques of FTIR, MS, and NMR. These compounds, confirmed to be oxysophocarpine, oxymatrine, matrine, and sophoridine, were then determined their therapeutic actions. The SF extracts and the compounds did not possess significant antioxidant activity using the DPPH and MDA assays, and cytotoxicity action using the MTT assay on the MDA-MB-231 breast cancer cell line. On the other hand, the SF total extract yielded a moderate acetylcholinesterase inhibition effect, with an IC50 of 0.1077 ± 0.0023 mg/mL. In summary, the SF extract demonstrated potential effects as an anti-acetylcholinesterase agent and could be further researched to become a pharmaceutical product for diseases related to acetylcholine deficiency, such as dementia.