Polo‐like kinase 1 (Plk1), a conserved Ser/Thr mitotic kinase, has been identified as a promising target for anticancer drug development because its overexpression is correlated with malignancy. Here, we found that genistein, an isoflavone, inhibits Plk1 kinase activity directly. Previously the mitotic disturbance phenomenon induced by treatment with genistein was not fully explained by its inhibitory effect on EGFR. In kinase profiling assays, it showed selectivity relative to a panel of kinases, including EGFR. Treatment with genistein induced cell death in a concentration‐dependent manner in cancer cells from diverse tissue origins, but not in non‐transformed cells such as hTERT‐RPE or MCF10A cells. We also observed that genistein tended to be more selective against cancer cells with mutations in the TP53 gene. TP53‐depeleted LNCaP and NCI‐H460 cells using shRNA targeting human TP53 were more sensitive to cell death by treatment of genistein. Furthermore, genistein induced mitotic arrest by inhibiting Plk1 activity and, consequently, led to mitotic catastrophe and apoptosis. These data suggest that genistein may be a promising anticancer drug candidate due to its inhibitory activity against Plk1 as well as EGFR and effectiveness toward cancer cells, especially those with p53‐mutation. J. Cell. Physiol. 232: 2818–2828, 2017. © 2016 Wiley Periodicals, Inc. The inhibitory activity of genistein on the Plk1 kinase was relatively selective to its activity against a panel of other kinases, including EGFR. Genistein had a cytotoxic effect in human cancer cells from diverse tissue origins, but not in non‐transformed cells. The sensitivity of cancer cells containing mutations in the TP53 gene to genistein tended to be higher when compared with that of cells with wild‐type TP53. Since around 50% of solid tumors have mutant TP53, the development of genistein as an anticancer drug would be valuable for treatment of such tumors.