The fungus Antrodia cinnamomea has been used as a folk medicine for various diseases, especially cancer. When A. cinnamomea is cultured on the original host, an endangered woody plant Cinnamomum kanehirai Hayata, the fungus produces more active ingredients, but its growth is slow. Here, C. kanehirai leaf ethanol extract (KLEE) was used as a substitute for C. kanehirai wood to culture A. cinnamomea on solid medium to shorten the culture period and produce active metabolites en masse. The antioxidant activities of methanol extracts from A. cinnamomea cultured on KLEE (MEAC-KLEE) were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect, reducing power, and ferrous ion-chelating effect, and the effective concentration (EC₅₀) values were 0.27, 0.74, and 0.37 mg mL⁻¹, respectively. MEAC-KLEE exhibited specific anti-proliferative activity against a non-small-cell lung cancer cell line (A549) by Annexin V assay. A secondary metabolite (2,4-dimethoxy-6-methylbenzene-1,3-diol, DMMB) present in the extract (MEAC-KLEE) was purified by high-performance liquid chromatography (HPLC) and identified by nuclear magnetic resonance (NMR) spectra. DMMB exhibited moderate antioxidant activity against DPPH radicals and reducing power, with EC₅₀ values of 12.97 and 25.59 μg mL⁻¹, respectively, and also induced apoptosis in A549 cells. Our results provide valuable insight into the development of DMMB for nutraceutical biotechnology.