Xanthium spinosum is a cosmopolitan annual herb used in traditional medicine worldwide. Although known from ethnobotanical studies, the species is scarcely investigated from the aspects of phytochemistry and biological activity. Therefore, the phenolic composition and biological activity of X. spinosum were examined. Plant specialised metabolites (phenolics) extracted from the roots, leaves and fruits with dichloromethane:methanol (1:1) were analysed by liquid chromatography mass spectrometry (LC-MS). In total 10 phenolic compounds were identified and quantified. Six compounds were common to all the extracts. Chlorogenic acid was the most abundant constituent in all the extracts (4.262 mg/g in the fruit extract, 0.820 mg/g in the leaf extract, and 0.540 mg/g in the root extract). The biological activity (antimicrobial and antibiofilm) of the extracts was tested against 12 microfungi and 12 bacterial strains by the microdilution method. All the extracts exhibited moderate antimicrobial and antibiofilm activity and inhibited the growth of most of the examined microorganisms. The obtained results indicate the potential role of the tested extracts in pharmacy and medicine.