Colorectal cancer is one of the most lethal cancers with a high mortality rate. Chemotherapy results in drug resistance in some cases; hence, herbal medicines are sometimes used in adjunct with it. Eugenol has been reported to have anti-inflammatory, antioxidant, and anticancer properties. Metabolomics is a study of metabolic changes within an organism using high-throughput technology. The purpose of this research was to investigate the anticancer effects of eugenol and variations in p53, KRAS, and APC gene expression and metabolic changes associated with the abovementioned gene expressions using 1HNMR spectroscopy. The MTT method was used to determine cell viability and its IC50 detected. After treating HT-29 cells with IC50 concentration of eugenol, RNA was extracted and cDNA was obtained from them and the expression of p53, KRAS, and APC genes was measured using the qRT-PCR technique. Metabolites were extracted using the chloroform-ethanol method, lyophilized, and sent for 1HNMR spectroscopy using the 1D-NOESY protocol. Chemometrics analysis such as PLS-DA was performed, and differentiated metabolites were identified using the Human Metabolome Database. Integrated metabolic analysis using the metabolites and gene expression was performed by the MetaboAnalyst website. The observed IC50 for eugenol was 500 μM, and the relative expression of APC and p53 genes in the treated cells increased compared to the control group, and the expression of KRAS oncogene gene decreased significantly. The crucial changes in convergent metabolic phenotype with genes were identified. The results indicate that eugenol exhibits its antitumor properties by targeting a specific biochemical pathway in the cell’s metabolome profile due to changes in genes involved in colon cancer.