Introduction: Breast cancer is the leading cause of cancer death in women all around the world. The research on new models, such as 3D cell systems, is ongoing, with the aim of developing effective anticancer drugs. One of them is benzimidazole, the core of which is a part of many therapeutic agents, and the mechanism of action directly focuses on tumor cells, thus favoring further research as potential cytostatic. Objectives: To investigate whether the antiproliferative potential of benzimidazole derivatives on the breast cancer cell lines differs in 2D and 3D cell cultures. To determine changes in the cell cycle. Materials and methods: The inhibitory effect of benzimidazole derivatives on MDA-MB-231 and MCF-7 tumor cell growth rates was determined by MTT test in 2D and 3D cultures. Changes in the cell cycle of the examined cell lines were monitored by flow cytometry. Results: Differences between the antiproliferative effects of benzimidazole derivatives on the cell growth in 2D culture compared to 3D culture were observed. The best growth inhibition of the cell growth resulted in the MB-129 derivative on both cell lines at 1x10-4 M concentration. Cell-specific changes in individual phases of the cell cycle were recorded; on MCF-7 cells, the MB-123 derivative caused their accumulation in the G1-phase, and the MDA-MB-231 cell line was the most important compound MB-129 leading to accumulation of cells in the S-phase of the cycle. Conclusion: Benzimidazole derivatives exhibit antiproliferative effects on the breast cancer cell growth, depending on the concentration applied and culturing conditions. MB-129 derivative had statistically most significant effect. Exposure of the cells to the investigated derivatives has led to changes in the cell cycle.