Purpose: The current study aimed to determine the phytochemicals and anti-breast cancer potential of Sphaeranthus indicus.Methods: S. indicus flowers were extracted with methanol followed by fractionation using n-hexane. For the chemical composition of n-hexane fraction, qualitative phytochemical and GC-MS analysis were performed. The anti-proliferative activity was measured by MTT assay, whereas, cytotoxic and proapoptotic effects in MCF-7 (breast cancer) cells were determined using propidium iodide, 4′,6-diamidino- 2-phenylindole, dichlorofluorescin diacetate, and JC-1 staining through fluorescent microscopy.Results: The phytochemical analysis indicated presence of phytosterols, oils and resins in the nhexane fraction. GC-MS analysis showed that n-hexane fraction comprises of 11 compounds including methyl esters of caprylic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid and behanic acid. The tested fraction showed remarkable cytotoxic activity against breast cancer (MCF-7) cells while it was found less toxic towards non-cancerous (BHK-21) cells. Furthermore, morphological assessment through fluorescent microscopy revealed cytotoxic and apoptotic effects by improved cell membrane permeability, increased reactive oxygen species level, compromised mitochondrial activity and condensation of chromatin network.Conclusion: The n-hexane fraction of S. indicus contains phytosterols, oils and fatty acid methyl esters and produced apoptotic effect against breast cancer cells.