Mitochondria are involved in many processes in eukaryotic cells. They play a central role in energy conservation and participate in cell metabolism and signaling pathways. Mitochondria are the main source of reactive oxygen species, excessive generation of which provokes numerous pathologies and cell death. One of the most promising approaches to the attenuation of oxidative stress in mitochondria is the use of targeted (i.e., transported exclusively into mitochondria) lipophilic cationic antioxidants. These compounds offer advantages over conventional water-soluble antioxidants because they induce the so-called "mild uncoupling" and can prevent collapse of the membrane potential in low, nontoxic concentrations. A novel mitochondria-targeted antioxidant, SkQT1, was synthesized and tested within the framework of the research project guided by V. P. Skulachev. The results of these experiments were initially reported in 2013; however, one publication was not able to accommodate all the data on the SkQT1 interactions with isolated mitochondria and cells. Here, we examined comparative effects of SkQT1 and SkQ1 on rat liver mitochondria (with broader spectrum of energy parameters being studied) and yeast cells. SkQT1 was found to be less effective uncoupler, depolarizing agent, inhibitor of respiration and ATP synthesis, and "opener" of a nonspecific pore compared to SkQ1. At the same time SkQ1 exhibited higher antioxidant activity. Both SkQT1 and SkQ1 prevented oxidative stress and mitochondria fragmentation in yeast cells exposed to t-butyl hydroperoxide and promoted cell survival, with SkQT1 being more efficient than SkQ1. Together with the results presented in 2013, our data suggest that SkQT1 is the most promising mitochondria-targeted antioxidant that can be used for preventing various pathologies associated with the oxidative stress in mitochondria.