The phytochemical, antiradical, and enzyme inhibition profile of three solvent extracts (ethyl acetate, methanol, water) of Origanum sipyleum were assessed. We also performed a pharmacological study in order to explore protective effects induced by extracts in inflamed colon. LC-MS analysis revealed that the extracts contained different classes of phenolics. The aqueous extract showed the highest antioxidant and acetylcholinesterase (AChE) inhibitory effects. Total phenol and flavonoid contents were highest in aqueous and ethyl acetate extract, respectively. All extracts were effective in reducing colon pro-oxidant and pro-inflammatory biomarkers. The extracts revealed also able to inhibit fungal and bacterial species involved in ulcerative colitis, including Candida albicans, Candida tropicalis, Staphylococcus aureus, and Staphylococcus thyphimurium. Finally, we also showed the antiproliferative effects exerted by the EA extracts on human colon cancer HCT116 cell line. Concluding, our results indicated that O. sipyleum extracts displayed promising therapeutic properties which warrants further validation. Practical applications: The present phytochemical and biological studies, including antioxidant, anti-inflammatory, and antimicrobic assessments, showed significant protective effects exerted by O. sipyleum extracts in an experimental model of ulcerative colitis. The results are intriguing and suggest potential applications O. sipyleum extracts as sources of natural agents for the management of clinical symptoms related to ulcerative colitis, characterized by increased burden of oxidative stress and microbiome dysbiosis.