In this study, cytotoxicity, anti-mildew and anti-wood-decay fungal activities of the bark essential oil and its constituents from Cunninghamia lanceolata var. konishii were evaluated in vitro against three human cancer cell lines, seven mildew fungi, and four wood decay fungi, respectively. The main compounds responsible for the cytotoxicity, anti-mildew, and anti-wood decay fungal activities were also identified. The essential oil from the barks of C. lanceolata var. konishii was isolated using hydrodistillation in a Clevenger-type apparatus, and characterized by GC-FID and GC-MS, respectively. The bark oil mainly consisted of ferruginol (15.1%), β-caryophyllene (11.2%), -cadinol (10.9%) and cedrol (10.1%). The essential oil exhibited cytotoxic activity against human oral, liver, and lung cancer cells. The active source compounds were -cadinol, cedrol, β-caryophyllene, and ferruginol. The bark oil was shown to have excellent anti-mildew and anti-wood-decay fungal activities, the active compounds being evaluated as -cadinol, cedrol, and ferruginol.