Xanthanoltrimer A–C (1–3), three xanthanolide sesquiterpene trimers, were first isolated from the fruits of Xanthium italicum Moretti by high-performance liquid chromatography-mass spectrometry-solid phase extraction-nuclear magnetic resonance spectroscopy (HPLC-MS-SPE-NMR). The structures of 1–3 were characterized by a series of spectroscopic methods, NMR calculations and electronic circular dichroism (ECD). Moreover, the cytotoxic, antiviral, anti-inflammatory, and antioxidant activities of 1–3 were screened, but they did not show significant biological activity.