Wasabi (Wasabia japonica (Miq.) Matsum.) is a pungent spice commonly consumed with sushi and sashimi. From the roots of this plant, a new 2-butenolide derivative (1) and 17 previously reported compounds (2–18) were isolated and structurally characterized. Their chemical structures were characterized based on the conventional NMR (¹H and ¹³C, COSY, HSQC, and HMBC) and HRESIMS data analysis. All of these phytochemicals (1–18) were evaluated for their antiproliferative effects on the four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and MKN-1), for their inhibitory activity on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 microglia cells, and for their nerve growth factor (NGF)-releasing effect from C6 glioma cells. Among the isolated compounds, compound 15 showed powerful antiproliferative activities against A549 and SK-MEL-2 cell lines with IC₅₀ values of 2.10 and 9.08 μM, respectively. Moreover, the new compound 1 exhibited moderate NO inhibition activity with IC₅₀ value of 45.3 μM.