A new enynyl-benzenoid, antrocamphin O (1,4,7-dimethoxy-5-methyl-6-(3'-methylbut-3-en-1-ynyl)benzo[d][1,3]dioxide), and the known benzenoids antrocamphin A and 7-dimethoxy-5-methyl-1,3-benzodioxole, were isolated from the fruiting bodies of Antrodia camphorata (Taiwanofungus camphoratus). The structure of antrocamphin O was unambiguously assigned by the analysis of spectral data (including 1D and 2D NMR, high-resolution MS, IR, and UV) and total synthesis. Compound 1 was prepared through the Sonogashira reaction of 5-iodo-4,7-dimethoxy-6-methylbenzene and 2-methylbut-1-en-3-yne as the key step. The benzenoids were tested for cytotoxicity against the HT29, HTC15, DLD-1, and COLO 205 colon cancer cell lines, and activities are reported herein.