Breast cancer ranks among the top three most common malignant neoplasms in Poland. The use of calcium ion-assisted electroporation is an alternative approach to the classic treatment of this disease. The studies conducted in recent years confirm the effectiveness of electroporation with calcium ions. Electroporation is a method that uses short electrical pulses to create transitional pores in the cell membrane to allow the penetration of certain drugs. The aim of this study was to investigate the antitumor effects of electroporation alone and calcium ion-assisted electroporation on human mammary adenocarcinoma cells that are sensitive (MCF-7/WT) and resistant to doxorubicin (MCF-7/DOX). The cell viability was assessed using independent tests: MTT and SRB. The type of cell death after the applied therapy was determined by TUNEL and flow cytometry (FACS) methods. The expression of Cav3.1 and Cav3.2 proteins of T-type voltage-gated calcium channels was assessed by immunocytochemistry, and changes in the morphology of CaEP-treated cells were visualized using a holotomographic microscope. The obtained results confirmed the effectiveness of the investigated therapeutic method. The results of the work constitute a good basis for planning research at the in vivo level and in the future to develop a more effective and safer method of breast cancer treatment for patients.