Mitochondria generate energy and building blocks required for cellular growth and function. The notion that mitochondria are not involved in the cancer growth has been challenged in recent years together with the emerging idea of mitochondria as a promising therapeutic target for oncologic diseases. Pentamethinium salts, cyan dyes with positively charged nitrogen on the benzothiazole or indole part of the molecule, were originally designed as mitochondrial probes. In this study, we show that pentamethinium salts have a strong effect on mitochondria, suppressing cancer cell proliferation and migration. This is likely linked to the strong inhibitory effect of the salts on dihydroorotate dehydrogenase (DHODH)-dependent respiration that has a key role in the de novo pyrimidine synthesis pathway. We also show that pentamethinium salts cause oxidative stress, redistribution of mitochondria, and a decrease in mitochondria mass. In conclusion, pentamethinium salts present novel anti-cancer agents worthy of further studies.