Antifungal activity of natural products is being studied widely. Saponins are known to be antifungal and antibacterial. We used bioassay-guided fractionation to have isolated eight steroid saponins from Tribulus terrestris L., which were identified as hecogenin-3-O-beta-D-glucopyranosyl (1-->4)-beta-D-galactopyranoside (TTS-8), tigogenin-3-O-beta-D-glucopyranosyl (1-->4)-beta-D-galactopyranoside (TTS-9), hecogenin-3-O-beta-D-glucopyranosyl (1-->2)-beta-D-glucopyranosyl (1-->4)-beta-D-galactopyranoside (TTS-10), hecogenin-3-O-beta-D-xylopyranosyl (1-->3)-beta-D-glucopyranosyl (1-->4)-beta-D-galactopyranoside (TTS-11), tigogenin-3-O-beta-D-xylopyranosyl (1-->2)-[beta-D-xylopyranosyl (1-->3)]-beta-D-glucopyranosyl (1-->4)-[alpha-L-rhamnopyranosyl (1-->2)]-beta-D-galactopyranoside (TTS-12), 3-O-[beta-D-xylopyranosyl (1-->2)-[beta-D-xylopyranosyl (1-->3)]-beta-D-glucopyranosyl (1-->4)-[alpha-L-rhamnopyranosyl (1-->2)]-beta-D-galactopyranosyl]-26-O-beta-D-glucopyranosyl-22-methoxy-(3beta,5alpha,25R)-furostan-3,26-diol (TTS-13), hecogenin-3-O-beta-D-glucopyranosyl (1-->2)-[beta-D-xylopyranosyl (1-->3)]-beta-D-glucopyranosyl (1-->4)-beta-D-galactopyranoside (TTS-14), tigogenin-3-O-beta-D-glucopyranosyl (1-->2)-[beta-D-xylopyranosyl (1-->3)]-beta-D-glucopyranosyl (1-->4)-beta-D-galactopyranoside (TTS-15). The in vitro antifungal activities of the eight saponins against five yeasts, Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Cryptococcus neoformans were studied using microbroth dilution assay. In vivo activity of TTS-12 in a Candida albicans vaginal infection model was studied in particular. The results showed that TTS-12 and TTS-15 were very effective against several pathogenic candidal species and Cryptococcus neoformans in vitro. It is noteworthy that TTS-12 and TTS-15 were very active against Candida albicans (MIC(80) = 10 and 2.3 microg/mL) and Cryptococcus neoformans (MIC(80) = 1.7 and 6.7 microg/mL). Phase contrast microscopy showed that TTS-12 inhibited hyphal formation, an important virulence factor of Candida albicans, and transmission electron microscopy showed that TTS-12 destroyed the cell membrane of Candida albicans. In conclusion, TTS-12 has significant in vitro and in vivo antifungal activity, weakening the virulence of Candida albicans and killing fungi through destroying the cell membrane.