Among known phenolic antioxidants, the overwhelming majority of compounds have lipophilic properties and the number of known water-soluble compounds is very small. The list of hydrophilic phenolic antioxidants can be expanded via the synthesis of a structurally related series of polyfunctional compounds for further research on their biological activity in vitro. New sulphur- and selenium-containing analogues of antioxidant potassium phenosan were synthesised. In vitro cytotoxicity and cytostaticity as well as antioxidant activity against H2O2-induced cytotoxicity to human cell lines (HepG2, Hep-2 and MCF-7) were investigated by high-content analysis. A selenium-containing analogue showed higher biological activity than did a sulphur-containing one. As compared to the activity of potassium phenosan, the selenium-containing analogue had a cell line-dependent antioxidant effect against H2O2-induced cytotoxicity: comparable in HepG2 cells and greater in Hep-2 cells. The selenium-containing analogue significantly increased the death of MCF-7 cells at concentrations above 50 µM. The sulphur-containing analogue has lower biological activity as compared to potassium phenosan and the selenium-containing analogue.