The study was designed to investigate the anticancer effect of phenazine-1-carboxamide (PCN) isolated from the bacterium Pantoea agglomerans naturally present in soil. PCN showed cytotoxicity in a dose-dependent manner, and inhibitory concentrations on the cancer cell lines A549, HeLa, and SW480 were between 32 and 40 μM. Significantly increased concentrations of lactate dehydrogenase were found with increasing concentrations of PCN, which resulted in increased destruction of the cancer cell membrane. A significantly increased p53 level was accompanied by the increased production of cytochrome c protein in all cancer cell lines studied. This condition in cells leads to the overexpression of caspase 3 and Bcl-2 family proteins. Upregulation and downregulation of proapoptotic and antiproapoptotic proteins were analyzed for their messenger RNA and protein expression. The activation of caspases and their cleavage compounds paves the way for the complete apoptosis process in cancer cells. We conclude that P. agglomerans-derived PCN acts as an effective anticancer drug or compound.