Ginkgo biloba, as a medicinal plant in both traditional and western medicine, emerged as a potential therapeutic agent for the management of a variety of diseases, but ginkgo biflavones (bilobetin, isoginkgetin, and ginkgetin) application in cancer therapy and underlying mechanisms of action remained elusive. In the present study, we identified ginkgo biflavones as potential p53 activators that could enhance p53 protein expression level by inhibiting MDM2 protein expression. At the same time, they induced cell death independent of p53 transcriptional activity. Moreover, ginkgetin was a standout among ginkgo biflavones that reduced the survival of HCT-116 cells by induction of apoptosis and G2/M phase arrest. Furthermore, ginkgo biflavones induced ROS generation significantly, which resulted in ferroptosis. Finally, we provide evidence that ginkgetin strengthened the antitumor effect of fluorouracil (5-FU) in the HCT-116 colon cancer xenograft model. To sum up, ginkgo biflavones represent a new class of p53 activator that depends on the p53 wild-type status and warrants further exploration as potential anticancer agents.