Abstract <p><b>Objective:</b> In the present work, we synthesized previously unknown 2-alkyl esters and amides of phenothiazine containing triphenylphosphonium (TPP⁺) and F16 mitochondriotropic groups in the alkyl chain and evaluated their effects on six tumor cell lines using the non-cancerous HEK293 cell line as control cells. In addition, we studied the effect of new phenothiazine derivatives on a number of basic functional parameters of mitochondria. <b>Methods:</b> TPP⁺-phenothiazine derivatives were prepared by alkylation of 10<i>H</i>-phenothiazine-2-carboxylic acid with alkyltriphenylphosphonium salts containing 3, 4, 6 or 8 methylene units in the alkyl chains or by Steglich amidation using TPP⁺-aminoalkanes derivatives. F16 analogues were prepared by conversion of the acid to bromoalkyl esters followed by reaction with 1<i>H</i>-indol-3-ylvinylpyridine (neutral precursor of F16). The structures of all products were confirmed by 1D (¹Н, ¹³С, APT) and 2D homo- (COSY, NOESY) NMR experiments and high-resolution mass spectrometry. The cytotoxic properties of the compounds were evaluated with the MTT assay. Effects of phenothiazine and its derivatives on mitochondrial membrane potential, parameters of respiration and oxidative phosphorylation of mitochondria and hydrogen peroxide production by mitochondria were evaluated on isolated rat liver mitochondria. <b>Results and discussion:</b> The new compounds showed pronounced cytotoxicity, especially towards A549 lung adenocarcinoma cells, whereas the parent 10<i>H</i>-phenothiazine-2-carboxylic acid was non-toxic and phenothiazine demonstrated some specificity of cytotoxic effect on HEK293, A549 and HepG2 cell lines at concentrations with IC₅₀ 79.7, 54.0, and 32.5 μM. Among the series, TPP⁺ esters were substantially more potent than the corresponding amides and F16 derivatives, and activity increased with linker length (<i>n</i> = 4 &lt; 6 &lt; 8). In experiments with isolated rat liver mitochondria, triphenylphosphonium salts of the phenothiazine alkyl esters were significantly inferior to phenothiazine and 10<i>H</i>-phenothiazine-2-carboxylic acid <b>I</b> in antioxidant activity with respect to H₂O₂ and induced mitochondrial dysfunctions, in particular they decreased the efficiency of oxidative phosphorylation and dissipated the membrane potential. These effects appear to be the main cause of the cytotoxic activity of the new phenothiazine derivatives. <b>Conclusions:</b> The new 2-alkyl esters and amides of phenothiazine, containing mitochondriotropic cationic groups in the alkyl chain showed significant cytotoxic activity on human tumour cell lines in comparison to non-toxic phenothiazine and 10<i>H</i>-phenothiazine-2-carboxylic acid. The incorporation of mitochondriotropic groups into the molecule of phenothiazine significantly reduced the antioxidant protection of the redox-active phenothiazine core, while enhancing protonophore effect promoting mitochondrial dysfunction in rat liver mitochondria</p>