Water-soluble fullerene derivatives are good candidates for various biological applications such as anticancer or antimicrobial therapy, cytoprotection, enzyme inhibition, and many others. Their toxicity, both in tissue culture and in vivo, is a critical characteristic for the development and restriction of these applications. The effects of six water-soluble cationic and anionic polysubstituted fullerene derivatives on cytochrome c oxidase activity in rat brain mitochondria and the possibility of cytochrome c binding were studied. We found that the ability of these fullerene derivatives to bind with cytochrome c oxidase and charged molecules like eosin Y strongly depends on their electrostatic charge. As was shown, the cationic fullerene derivative inhibits cytochrome c oxidase that has the overall negative electrostatic potential completely, unlike anionic derivatives. Thus, it confirms the essential role of electrostatic interactions in the interaction of fullerene derivatives with the active site of enzymes. The results explore how cationic fullerene derivatives play a role in mitochondrial dysfunction, oxidative stress, and cytotoxicity.