Elicitation of the whole plant/seedling using a biotic perturbations (e.g. CO2) could be practical for strategic bio farming of plant-derived metabolites as elicitation modulates plant secondary metabolism that is often associated with the altered chemical constituents and regulation of other metabolic pathways. Labisia pumila is a popular herb known to contain high-value metabolic compounds that are able to scavenge free radicals associated with chronic diseases (e.g. cardiovascular disease, diabetes, several types of cancer). The obtained results revealed that carbon dioxide levels had a significant (P≤0.01) impact on the production of total phenolic, flavonoid and saponin contents. HPLC analysis showed, increasing the CO2 concentration from 400 to 1200μmolmol−1 resulted in enhanced gallic acid, pyrogallol, epicatecin, naringin, diadezin, quercetin and myricetin contents in the leaves of Labisia pumila var alata. On the other hand the GC and GC–MS identify and reproduce of new compounds under different level of CO2. Furthermore, the antioxidant potentials using DPPH, FRAP and NO2 scavenging of the leaf extract under 1200μmolmol−1 had 7.77%, 8.36% and 5.53% increased compared to control group. Finally, the anticancer activities demonstrated that leaf extracts grown under elevated CO2 concentration exhibited the strongest cytotoxicity effect towards cancer cells. These research indicated that CO2 enrichment result in improved secondary metabolites production and enhanced biological activities such as antioxidant and anticancer properties. This may present a useful agro technological tool of novel cultivation practice for producing medicinal herbs of improved quality and production of valuable metabolites.