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The fungus <i>Antrodia cinnamomea</i> has been used as a folk medicine for various diseases, especially cancer. When <i>A. cinnamomea</i> is cultured on the original host, an endangered woody plant <i>Cinnamomum kanehirai</i> Hayata, the fungus produces more active ingredients, but its growth is slow. Here, <i>C. kanehirai</i> leaf ethanol extract (KLEE) was used as a substitute for <i>C. kanehirai</i> wood to culture <i>A. cinnamomea</i> on solid medium to shorten the culture period and produce active metabolites en masse. The antioxidant activities of methanol extracts from <i>A. cinnamomea</i> 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.