Breast cancer remains a global challenge, with rising incidence rates. While treatment advancements have improved outcomes, systemic administration of cytostatic agents continues to cause severe adverse effects, including myelosuppression, heart failure, and infertility, due to non-specific biodistribution. Nanoparticle-based drug delivery systems have been explored as an alternative approach to improve therapeutics delivery and reduce non-specific effects. Zinc-based nanoparticles demonstrate ability to induce cytotoxic responses in cancer cells in vitro, making them suitable systems for probing cell-type-specific effects. In this study, we evaluated the in vitro cytotoxic effects of spherical zinc peroxide (ZnO₂) nanoparticles (20–80 nm and 50–300 nm), commercial ZnO nanoparticles, and tetrapodal ZnO (T-ZnO) microparticles in MCF-7 breast cancer cells and RMF-EG fibroblasts. ZnO₂ nanoparticles demonstrated dose-dependent cytotoxicity (1 µg/mL–10 mg/mL), inducing a significantly higher death rate of cancer cells than normal fibroblasts, which retained >75% viability at comparable doses. Flow cytometry investigations revealed that ZnO₂ nanoparticles exhibited preferential cellular uptake in MCF-7 cells as compared to fibroblasts. Overall, these findings indicate different cytotoxic responses of ZnO-based micro and nanoparticles between MCF-7 cells and RMF-EG fibroblasts under in vitro conditions. Further studies are required to validate these observations in more complex biological systems and to clarify the underlying mechanisms.