Introduction: Benzimidazole is a heterocyclic aromatic organic compound which is produced by fusion of benzene and imidazole. Because of its structure, it is similar to biomolecules which normally participate in cell processes and it exhibits an ability to attach to double-stranded DNA. Adding different bioactive functional groups to the molecule base increases the pharmacological properties of such molecules. For that reason, they are utilised as a base for development of new molecules with anti cancer activity. Objective: The aim of the study is to examine the effect of benzimidazole derivatives on the growth of cells under in vitro conditions and to determine if there is a difference in the effect of compounds on cells which grow in monolayer and spheroids. Materials and Methods: For the purposes of the research, two-dimensional and three-dimensional HeLa cell cultures were prepared. Cell lines were incubated at a temperature of 37 °C with 5 % CO2. After treating the cells with the benzimidazole derivatives (MB-123, MB-127, MB-129) and incubating them for 72 hours, the effect of tested compounds on cell growth was determined by an MTT assay. Results: Tested benzimidazole derivatives show cytotoxic activity dependent on the prepared concentration and cell culture. The best inhibitory effect on cell growth was detected for MB-127 at a concentration of 10-4 mol/L. Conclusion: Cells grown in 2D and 3D culture respond differently to the effects of benzimidazole derivatives depending on the type of compound and prepared concentrations.