In the present work, fruits of Prangos platychlaena Boiss. ex Tchihat. ssp. platychlaena (Apiaceae) collected from eastern (sample A) and central (sample B) parts of Turkey were subjected to hydrodistillation to yield essential oils (EOs). The chemical composition of P. platychlaena ssp. platychlaena fruit oil was analyzed by gas chromatography–flame ionization detector (GC–FID) and gas chromatography–mass spectrometry (GC–MS) techniques. An unknown compound with m/z 178 [M]+ in high percentages was detected in both EOs (45.8% and 11.2% in samples A and B, respectively). Column chromatography on silica gel was subsequently followed by the sample A and yielded a new acetylenic derivative (1), which was characterized by spectroscopic techniques as (2S)- 3,5-nonadiyne-2-yl acetate. Samples A and B oils were characterized by a series of acetylenic derivatives like 3,5-nonadiyne (24.5% and 5.8% in A and B, respectively), (Z)-3,5-nonadiyne-7-ene (0.2% in A), and (E)-3,5-nonadiyne-7-ene (0.5% in A). Monoterpenes α-pinene (6.8% and 12.8%), α-phellandrene (0.1% and 17.1%), and β-phellandrene (4.2% and 22.4%) were found to be the major components in P. platychlaena ssp. platychlaena EOs in samples A and B, respectively. The components of P. platychlaena ssp. platychlaena EOs were separated on the overpressured layer chromatography (OPLC) plates, and the plates were subsequently subjected to direct-bioautography assays using three plant pathogens such as Colletotrichum acutatum, C. fragariae, and C. gloeosporioides; however, no antifungal activity was observed. Due to a high yield, sample A was evaluated for its repellent activity against female mosquito Aedes aegypti and attraction of sterile male Mediterranean fruit fly Ceratitis capitata. Sample A demonstrated good repellency against Ae. aegypti in human-based cloth patch bioassays and no attraction to C. capitata in short range bioassays.