Cyclohexyl thiosemicarbazone derivatives (C1-14) were synthesized, characterized and evaluated against HER-2 over expressed breast cancer cells. The synthesized compounds were screened in vitro against four breast cancer cell lines; SKBr-3, MCF-7, MDA-MB-468 and MDA-MB-231. All the compounds showed activity against HER-2 over expressed SKBr-3 cells with (IC₅₀ = 25.6 ± 0.07 μM-61.6 ± 0.4 μM). The most active compounds inhibit ALDH⁺ breast cancer stem cells more effectively than the cancer stem cells specific agent Salinomycin. Immunohistochemistry staining also confirmed that these compounds inhibit the expression of HER-2 on SKBr-3 cells. Compound C2 significantly inhibited the cell migration and cell adhesion of breast cancer cell lines. Compound C2 was found to most active compound of this series targeting HER-2 over expressed breast cancer cells.