Cancer is one of the main causes of death worldwide. Platinum complexes (i.e., cisplatin, carboplatin, and others) are currently heavily used for the treatment of different types of cancer, but unwanted effects occur. Ruthenium complexes have been shown to be potential promising alternatives to these metal-based drugs. In this work, we performed a structure-activity relationship (SAR) study on two small series of Ru(II) polypyridyl complexes of the type [Ru(L1)2(O^O)]Cln (3-8), where L1 is 4,7-diphenyl-1,10-phenantroline (DIP) or 1,10phenantroline (phen), and O^O is a symmetrical anionic dioxo ligand: oxalate (ox, n = 0), malonate (mal, n = 0), or acetylacetonate (acac, n = 1). These two self-consistent series of compounds allowed us to perform a systematic investigation for establishing how the nature of the ligands and the charge affect the anticancer properties of the complexes. Cytotoxicity tests on different cell lines demonstrated that some of the six compounds 3-8 have a promising anticancer activity. More specifically, the cationic complex [Ru(DIP)2(η 2-acac)]Cl (4) has IC50 values in the mid-nanomolar concentration range, lower than those of cisplatin on the same cell lines. Interestingly, [Ru(DIP)2(η 2-acac)]Cl was found to localize mainly in the mitochondria, whereas a smaller fraction was detected in the nucleus. Overall, our SAR investigation demonstrates the importance of combining the positive charge of the complex with the highly lipophilic diimine ligand DIP.