Tempe is a soy fermentation product and has been associated with many health benefits. It is usually consumed after 48 hours of fermentation; however extended fermentation is sometimes favoured. Since cooking involves water, it is of interest to study the health benefits of tempe water extract. In this study, we focused our study on total phenols, total flavonoids, DPPH free radical scavenging activity, cytotoxic activity, and identification of the main flavonoids. Our findings demonstrated that concentration of water-soluble flavonoids in tempe increased as fermentation time was prolonged. Fermentation for 60 hr resulted in extracts with very noticeable cytotoxic activity against MCF-7 cancer cell line (IC50 of 8.70 µg/ml). The cytotoxic activity of the extracts was fermentation time-dependent following non-linear relationship. The activity showed strong inverse correlation with total phenols and flavonoids in the extracts. LC-MS/MS data suggested that extracts from control and 24-hr fermented tempe demonstrated the highest responses for genistein, genistin, daidzein, daidzin, and glycitein, compared to those of other treatments and therefore suggested that excessive amount of the isoflavones, particularly genistein, induced cancer cell proliferation but lesser yet optimum concentration (observed at 60 hr-fermentation) contributed to otherwise effect.