By inspiration of significant anticancer activity of our previously screened natural ergosterol peroxide (1), a series of novel ergosterol peroxide 3-carbamate derivatives were synthesized and characterized. The anti-proliferative activity of synthesized compounds against human hepatocellular carcinoma cells (HepG2, SK-Hep1) and human breast cancer cells (MCF-7, MDA-MB231) were investigated. Compound 3d, 3f and their hydrochloride exhibited significant in vitro anti-proliferative activity against the tested tumor cell lines, with IC50 values ranging from 0.85 to 4.62 μM. Furthermore, fluorescence mitochondria-targeting images showed that the designed coumarin-3d conjugate (5) localized mainly in mitochondria, leading to enhanced anticancer activities over the parent structure (1). As a whole, it appeared that substituent changes to the C-3 position could serve as a promising launch point for further design of this type of steroidal anticancer agents.