As a part of a broad screening of antifungal agents from plant origin, crude extracts from Panamanian plants having related types of constituents displayed significant activities in an agar overlay thin layer chromatography assay against a susceptible strain of Candida albicans. These were the methanolic extract of the leaves of Schefflera systyla and Odontadenia puncticulosa and of the stems of Conostegia speciosa, that are species not previously investigated from a phytochemical viewpoint. For all plants, high-performance liquid chromatography (HPLC) antifungal activity based profiling allowed the rapid localization of antifungal agents that were further obtained by targeted isolation procedure by semi-preparative HPLC or medium pressure liquid chromatography (MPLC) after LC gradient transfer. Different hederagenin saponins and one aglycone were found to be responsible for the antifungal activities of the extracts. Alpha-hederin was the antifungal of S. systyla, pulsatilla saponin D and 3 eta-O-[beta-D-xylopyranosyl-(1 -> 3)-alpha-L-rhamnopyranosyl-(1. 2)-[beta-D-glucopyranosyl-(1 -> 4)]-alpha-L-arabinopyranosylhederagenin of O. puncticulosa and arjunolic acid of C. speciosa. Their minimal inhibitory concentration (MIC) against planktonic and biofilm cells of C. albicans were determined. Alpha-hederin was the most potent compound with a MIC of 4 mu g mL(-1). Structurally related compounds (hederagenin, medicagenic acid 3-O-beta-D-glucopyranoside and medicagenic acid) were used as standards and tested for comparison purposes. In order to better estimate the potential of these triterpenoids as antifungal agents, their cytological effects on C. albicans were determined by transmission electron microscopy (TEM) and the in vivo activity of alpha-hederin, medicagenic acid 3-O-beta-D-glucopyranoside and medicagenic acid was evaluated for the first time in the Galleria mellonella larvae model.