In natural products research, the efficient purification of molecules from large amount of complex extracts is a key element. In this regard, an integrative strategy for efficient mass spectrometry (MS)-guided isolation of antifungal compounds has been developed. First, at-line HPLC antifungal activity based profiling and HPLC-PDA-MS profiling were used to localise the compounds of interest on the analytical scale. Then, the analytical gradient was geometrically transferred to the flash chromatographic level. Finally, an MS-triggered isolation of the localized bioactive molecules was realized using high resolution flash chromatographic columns (15 µm spherical particles) coupled to a single quadrupole mass spectrometer via a splitter system. This isolation strategy was applied for the MS-targeted purification of the antifungal principles from the Chinese liverwort Chiloscyphus polyanthos (L.) Cord. This rational methodology has a high potential for the targeted large-scale purification of bioactive compounds avoiding the need to repeat a given bioassay at each isolation step. Seven sesquiterpene lactones were isolated, of which five were found bioactive and one was reported as a new compound. The absolute configuration of some compounds was established for the first time by electronic circular dichroism (ECD) spectroscopy.