Pepolo (Bischofia javanica Blume) is an Indonesian native plant whose stem bark is widely used as a traditional medicine for various diseases. Therefore, this study aims to determine the chemical profile of the secondary metabolites of the Pepolo stem bark (PSB) extract and its effectiveness as a potential immunomodulatory and anticancer agent. The PSBs were extracted by a reflux technique with 96% v/v ethanol as solvent. The immunomodulatory activity of the PSB extract was evaluated based on its effect on the macrophages of BALB/C mice, while the cytotoxicity and anticancer potential were observed as the PSB extract response on brine shrimp lethality test and MCF-7 cancer cells, respectively. The chemical profiles of the secondary metabolites were analyzed using a Liquid Chromatography-Mass Spectrometry/Mass Spectrometry instrument. The result is that the PSB extract showed its potential as an immunomodulatory and anticancer agent. The toxicity evaluation showed that the PSB extract was classified as a toxic material. More importantly, the PSB extract showed an activity against MCF-7 cancer with lethal concentration 50% (LC 50 ) values of 68.46 µg/mL and an inhibition concentration 50% (IC 50 ) of 362.36 µg/mL, respectively. The chemical evaluation suggested that the terpenoid and polyphenolic compounds are responsible for the immunomodulatory and anticancer mechanisms. In summary, our work showed that the PSB extract is a potential alternative source for the treatment of breast cancer and supports the immune response.