Trigonella foenum-graecum, commonly known as fenugreek, is widely used as a spice in India, Pakistan, and China for centuries. It has been suggested that biotransformation of the glycosides by various microorganisms increase the biological activity and bioavailability. This study aims to characterize the biotransformation of the fenugreek saponins by food microorganisms and to assess the cytotoxicity of the biotransformed fenugreek extract and dioscin which is a compound produced during biotransformation against various cell lines. The production of dioscin was confirmed by TLC and LC-MS analysis. The cytotoxicities against HT-29 colon cell line were increased after biotransformation by Aspergillus usamii, Bifidobacterium infantis, Bifidobacterium sp. Int57, and Leuconostoc paramesenteroides but decreased by Aspergillus niger. The $IC_{50}$ values of dioscin against five different tumor cell line cells ranged 0.72-3.22 and $2.88-8.53;{mu}M$ in 2% and 10%(v/v) FBS containing media, respectively. Real time cell analysis showed that HT-29 cells died immediately after treatment with dioscin above $7.5;{mu}M$. Dioscin at $5;{mu}M$ increased sub G1 phase cells when assessed with flow cytometric analysis. Since caspase-3 activity was increased by dioscin, cytotoxicity of dioscin might be related to apoptosis. Taken together, the structure and the level of saponin components in fenugreek were differentially affected by various food microorganisms. The safety and the cytotoxicity of dioscin produced during biotransformation need to be further evaluated in vivo.