Colorectal cancer remains a leading cause of cancer-related mortality, necessitating the development of novel therapeutic strategies. In this study, we investigate the pharmacological and toxicological properties of JAMC-4/108, a traditional Mongolian herbal extract, in human colonic adenocarcinoma (HT-29) and normal colonic epithelial (CCD 841 CoTr) cells. Using high-performance liquid chromatography–electrospray ionization–quadrupole time-of-flight–tandem mass spectrometry fingerprinting, we characterized its chemical composition, while a combination of cellular, biochemical and molecular assays provided mechanistic insights into its cytotoxic effects. The ethanol extract of JAMC-4/108 exhibited substantial selective cytotoxicity in HT-29 cells by inducing apoptosis and necrosis via caspase-3, cleaved caspase-7 and poly(ADP-ribose) polymerase activation, while the water extract primarily triggered caspase-9–mediated apoptosis. Both extracts modulated oxidative stress pathways, increasing Nrf2, Keap1 and LC3A/B levels, with the ethanol extract also upregulating NQO1, suggesting metabolic implications. In contrast, the ethanol extract had minimal apoptotic effects in normal colonic epithelial cells, whereas the water extract primarily influenced caspase-9 expression, indicating a distinct toxicity profile. Additionally, both extracts altered cell cycle progression–stimulated NOx release and modulated Fe3+ ion and DPPH radical pools in a concentration-dependent manner. Our findings highlight the selective anticancer potential of JAMC-4/108 and provide mechanistic insights into its pharmacological and toxicological effects, supporting its further evaluation for therapeutic development and safety assessment.