Medicinal plants belonging to the genus Ardisia are traditionally used to cure various human diseases including inflammation and cancer. This study aimed to purify and characterize cytotoxic and anti-inflammatory compounds from Ardisia sieboldii leaves. Bioassay-guided chromatographic analyses yielded three compounds, 2-methyl-5-(8Z-heptadecenyl) resorcinol (1), 5-(8Z-heptadecenyl) resorcinol (2), and ardisiaquinone A (3), whereas liquid chromatography-electrospray ionisation-mass spectrometry chemical profiling revealed the presence of diverse resorcinol and alkylbenzoquinone derivatives in cytotoxic 70% methanol extracts. Chemical structures of 1-3 were confirmed by spectroscopic methods including 1H NMR (nuclear magnetic resonance), 13C NMR, and electrospray ionisation-mass spectrometry. Compounds 1 and 2 were purified from A. sieboldii for the first time, and all three compounds showed cytotoxicity against a panel of cancer cell lines and brine shrimps in a dose-response manner. Among them, compound 2 exhibited the highest cytotoxicity on cancer cells (IC50 values of 8.8-25.7 μM) as well as on brine shrimps (IC50 value of 5.1 μM). Compounds 1-3 exhibited anti-inflammatory effects through inhibiting protein denaturation (IC50 values of 5.8-9.6 μM), cyclooxygenase-2 activity (IC50 values of 34.5-60.1 μM), and nitrite formation in RAW 264.7 cells. Cytotoxic and anti-inflammatory activities of 1-3 demonstrated in this study deserve further investigation for considering their suitability as candidates or leads to develop anticancer and anti-inflammatory drugs.