Background Er-Miao-San (EMS) is a classic prescription in traditional Chinese medicine (TCM) for the treatment of colorectal cancer (CRC) and has shown promising therapeutic effects in clinical practice. However, the specific components and molecular mechanisms of EMS remain unclear. Purpose The aim of this study was to analyze the effective components and molecular mechanisms of EMS in treating CRC through network pharmacology techniques and experimental validation. Methods The Traditional Chinese Medicine Systems Pharmacology database was used to screen the main active chemical components and targets of the EMS formula. The compound structures were verified using the PubChem database, which is an organic small-molecule bioactivity database. GeneCards and OMIM databases were utilized to predict target genes related to CRC. The Cytoscape 3.8 software was used to construct a “Drug-Active Ingredient-Target-Disease” intersection network. The STRING database was employed to analyze the core target protein–protein interaction network shared by EMS and CRC. The core targets were further subjected to Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis using the R language. Molecular docking between the core targets and the major active chemical components of EMS was performed using AutoDock software. The impact of the core targets on the prognosis of CRC patients was analyzed using the R language. Subsequently, we validated the potential mechanisms predicted by network pharmacology for the inhibition of CRC cell proliferation by the key proteins in the relevant pathways through CCK8 cell proliferation assays and Western blot experiments. Results Molecular docking results showed good docking affinity between the key active components, such as quercetin and baicalein, of EMS and the core targets. Kaplan–Meier survival analysis demonstrated a close correlation between the core targets and the survival prognosis of CRC patients. Cellular experiments showed that EMS significantly inhibited the proliferation of CRC cells and may promote apoptosis and autophagy of CRC cells by suppressing the expression of key proteins in the PI3K/AKT pathway. Conclusion The TCM formula EMS exerts anti-CRC effects through multiple pathways and targets, improving the prognosis and extending the survival period of CRC patients. This study provides preliminary insights into the effective components and molecular mechanisms of EMS in the treatment of CRC, which were preliminarily validated through molecular docking and experimental approaches.