Low therapeutic efficacy and resistance to conventional drugs remain the major obstacles in modern chemotherapy. Scientists around the world are therefore constantly searching for new drugs with different modes of action that, alone or in combination with other drugs, can help cancer patients. In recent years, selenium-containing compounds have been extensively studied as anticancer agents due to the characteristic properties of selenium. The main aim of this study was to evaluate the chemotherapeutic potential of isoselenazolium salts, a novel class of organoselenium compounds, and to explore their mechanism of action. The first series of isoselenazolium salts exhibited selective cytotoxicity against breast cancer cell lines. These compounds modulated mitochondrial respiration, increased reactive oxygen species production, altered NAD+ homeostasis, inhibited poly(ADP-ribose) polymerase 1 (PARP1) and possibly interfered with cardiolipin, a signature phospholipid of mitochondria. To test the latter, a new fluorescent cardiolipin-specific probe and a competitive binding assay were developed to quantitatively describe the drugs` affinity for cardiolipin. Using a new method, it was proved that the affinity of isoselenazolium salts for cardiolipin is low, and their interaction is not related to the pharmacological effect. The second series of isoselenazolium salts with a modified structure was designed to have improved PARP1 inhibitory activity. These compounds displayed high cytotoxicity against T-cell leukaemia, breast, liver, and lung cancer cells. Although the PARP1 inhibitory activity was improved, detailed studies of the mechanism of action showed that isoselenazolium salts are potent and selective inhibitors of pyruvate kinase M2 (PKM2), an enzyme, that is highly expressed in various types of tumours. On the basis of NMR, size-exclusion chromatography, mass photometry, differential scanning fluorimetry, isothermal titration calorimetry, and enzyme kinetic data, isoselenazolium salts were found to be competitive inhibitors and at the same time induce an unstable PKM2 homotetramer formation. Thus, PKM2 translocation to the nucleus is blocked, preventing its nonmetabolic functions. The discovery of robust PKM2 inhibitors could serve as the basis for new anticancer drug candidates and provide an important insight into the fundamental role of PKM2 in oncogenesis. 3.1 Basic medicine; Sub-Sector - Pharmaceutical Pharmacology. Keywords: cancer, cardiolipin, isoselenazolium salts, metabolic reprogramming, PARP1, pyruvate kinase M2, selenium. This work was supported by ERDF “Development of a novel potent PARP inhibitor” (No. 1.1.1.1/19/A/016) and BioMedPharm (No. VPP-EM-BIOMEDICINA-2022/1-0001) projects.