The present study aimed to discover anti-inflammatory molecules from Dendrobium officinale using mass spectrometry-based molecular networking technology and evaluate its therapeutic potential against ulcerative colitis (UC). We employed activity-labeled molecular networking (ALMN) to identify anti-inflammatory molecules from D. officinale extracts. From 3700 metabolite molecules, five top anti-inflammatory molecules were prioritized and isovitexin was further selected to validate its alleviation effect on a mouse model of DSS-induced UC. Mechanistically, isovitexin was found to restore intestinal epithelial barrier function and modulate autophagy via suppression of the PI3K/AKT signaling pathway. Additionally, isovitexin treatment altered the diversity and composition of the gut microbiota, enriching beneficial bacteria and reducing pathogenic bacteria. This was accompanied by changes in short-chain fatty acid levels, particularly the increase of butyric and valeric acids. Our study provided insights into the pharmacological actions of D. officinale and highlighted isovitexin as a novel candidate for UC treatment.