Gas chromatographic analysis revealed that the oil of Crinitaria tatarica was rich in sabinene (32.1%), β-pinene (8.8%), and two unknown (M+200) compounds (I) and (II) (21.4% and 3.4%). One-step multiple fractionation of the oil and separation of two unknown constituents were performed using preparative capillary gas chromatography connected to preparative fraction collector system. This combination allowed separation and recover of sufficient quantities of two unknown compounds with high purity from complex oil matrix. Separation conditions (column temperature, cooling temperature, flow rate, injection volume, cut time) were optimized to achieve the best isolation and successful collection. The target compounds were separated from the oil using a HP Innowax (Walt & Jennings Scientific, Wilmington, DE, USA) preparative capillary column in rapid one-step manner with 95.0% purity. Trapping of the isolated compounds in collector system was facilitated by cooling with liquid nitrogen. Structure determination was accomplished by spectral analysis including ultraviolet, nuclear magnetic rezonance, and high-resolution electrospray ionization mass spectrometry. Z- (I) and E-artemidin (II) were isolated for the first time from this species. Crinitaria tatarica oil and Z- (I) and E-artemidin (II) were evaluated for biological activity.