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An investigation of the essential oil (EO) and the crude MeOH extract of a Bhutanese variety of Ajania nubigenausing GC/GC-MS and NMR found the following: a) one kg of the dried plant material contained 0.7% w/w EO; b) 44 of the 53 GC-FID peaks of the EO were identified with (3R,6R)-linalool oxide acetate (75.8 %) as the major constituent (chemotype II) and chamazulene as a new sub-chemotype; c) purification of the EO furnished (3R, 6R)-linalool oxide acetate (1), chamazulene (2), (E)-2-(2,4-hexadiynylidene)-1,6-dioxaspiro[4,4]non-3-ene (3), and (Z)-2-(2,4-hexadinylidene)-1,6-dioxaspiro[4,4]non-3-ene (4); d) from the crude MeOH extract, four flavonoid compounds: 1-(4-hydroxyphenyl)propan-1-one (5), oxyanin B (6), luteolin (7) (major) and the luteolin-7-O-β-D-glucoside (8) were isolated; e) among the EO and pure compounds tested for biological activities, compound 7exhibited a broad range of moderate antiplasmodial, cytoxicity and antimicrobial activities; c) compound 8showed significant in vitroantiplasmodial activity against P. falciparumstrains TM4/8.2 and K1CB1 (multidrug resistant strain) and was identified as a potential antimalarial scaffold; and f) the in vitroantimicrobial and cytotoxicity activities were in alignment with the traditional medical uses of this plant and thus substantiate its use in Bhutanese traditional medicine.