BACKGROUND Malancao (MLC) is a traditional Chinese medicine with a long history of utilization in treating ulcerative colitis (UC). Nevertheless, the precise molecular mechanisms underlying its efficacy remain elusive. This study leveraged ultra-high-performance liquid chromatography coupled with exactive mass spectrometry (UHPLC-QE-MS), network pharmacology, molecular docking (MD), and gene microarray analysis to discern the bioactive constituents and the potential mechanism of action of MLC in UC management. AIM To determine the ingredients related to MLC for treatment of UC using multiple databases to obtain potential targets for fishing. METHODS This research employs UHPLC-QE-MS for the identification of bioactive compounds present in MLC plant samples. Furthermore, the study integrates the identified MLC compound-related targets with publicly available databases to elucidate common drug disease targets. Additionally, the R programming language is utilized to predict the central targets and molecular pathways that MLC may impact in the treatment of UC. Finally, MD are conducted using AutoDock Vina software to assess the affinity of bioactive components to the main targets and confirm their therapeutic potential. RESULTS Firstly, through a comprehensive analysis of UHPLC-QE-MS data and public database resources, we identified 146 drug-disease cross targets related to 11 bioactive components. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis highlighted that common disease drug targets are primarily involved in oxidative stress management, lipid metabolism, atherosclerosis, and other processes. They also affect AGE-RAGE and apoptosis signaling pathways. Secondly, by analyzing the differences in diseases, we identified key research targets. These core targets are related to 11 active substances, including active ingredients such as quercetin and luteolin. Finally, MD analysis revealed the stability of compound-protein binding, particularly between JUN-Luteolin, JUN-Quercetin, HSP90AA1-Wogonin, and HSP90AA1-Rhein. Therefore, this suggests that MLC may help alleviate intestinal inflammation in UC, restore abnormal lipid accumulation, and regulate the expression levels of core proteins in the intestine. CONCLUSION The utilization of MLC has demonstrated notable therapeutic efficacy in the management of UC by means of the compound target interaction pathway. The amalgamation of botanical resources, metabolomics, natural products, MD, and gene chip technology presents a propitious methodology for investigating therapeutic targets of herbal medicines and discerning novel bioactive constituents.