This study investigated the chemical composition, and antimicrobial activity against food-borne pathogens of the essential oil isolated from the heartwood of Calocedrus formosana from Taiwan. The oil, isolated by hydrodistillation in a Clevenger-type apparatus, was characterized using GC–FID and GC–MS. The major constituents were τ-muurolol (16.1%), α-cadinol (11.1%), α-terpineol (10.6%), thymol (8.5%), and β-thujaplicin (4.5%). The oil demonstrated strong activity against food-borne bacterial and fungal pathogens, and, to determine the source compounds responsible for this activity, the main components were individually evaluated. The most active source compounds were determined to be τ-muurolol, α-cadinol, thymol, and β-thujaplicin.