Cupressus is a genus of considerable medicinal value worldwide. The present work aims at assessing essential oil compositional differences within Cupressus in the context of its different genotypes and organs. Volatiles profiling was determined via gas chromatography-mass spectrometry coupled to chemometrics. A total of 75 volatile components were identified. Monoterpene hydrocarbons amounted for the major volatile class, with Î±-pinene and 3-carene most abundant in Cupressus sempervirens versus sabinene and terpinen-4-ol in Cupressus macrocarpa. Multivariate data analyses revealed that Î±-pinene, sabinene, terpinen-4-ol and 3-carene are the most significant for species classification. The five examined essential oils exhibited potential insecticidal and anticholinesterase effects, with C. sempervirens var. pyramidalis cone oil being the most potent for acetylcholinesterase inhibitory effect. The oil also exhibited potential fumigant toxicity toward Sitophilus oryzae and Tribolium castaneum. The results of the orthogonal partial least-square discriminant analysis revealed that Î±-pinene is the key bioactive component in Cupressus oil.